Quality Characteristics and Functional Properties of Paneer Fortified with Sesbania grandiflora Leaf Powder

Abstract

Sesbania grandiflora (Agathi) is a nutritionally and pharmacologically rich tropical legume tree whose leaves contain substantial quantities of phenolic acids, flavonoids, carotenoids, calcium, and iron. Despite their well-documented bioactivity, comprehensive multi-parameter characterisation of paneer fortified with S. grandiflora leaf powder has not previously been reported, representing the focus of the present study; paneer is the most widely consumed “acid–heat coagulated” dairy product across South Asia. The present study developed and characterised paneer fortified with S. grandiflora leaf powder at 0% (T1, control), 0.5% (T2), 1.0% (T3), and 1.5% (T4) and comprehensively evaluated the effects on proximate composition, pH, instrumental colour (CIE L*, a*, b*), total phenolic content (TPC), flavonoid content, antioxidant activity (phosphomolybdenum and DPPH methods), texture profile analysis (TPA), water activity (aw), HPLC-based phenolic profiling, “SDS PAGE” protein characterisation, microbiological quality, and sensory acceptability. Fortification significantly increased protein (19.00–22.00%), crude fibre (0.40–2.30%), ash (1.20–1.30%), TPC (2.18–5.82 mg GAE/g dw), flavonoid content (0.31–1.56 mg RE/g dw), and antioxidant activity (phosphomolybdenum assay: 0.03–0.16 mg AAE/g; p < 0.05) with increasing leaf powder levels, while fat and moisture declined proportionally. HPLC profiling identified quercetin and kaempferol as the dominant flavonoids in fortified treatments, with total quantified polyphenols reported at 3.35 mg/g dry weight in T4. TPA revealed that T3 achieved comparable hardness to T1 (58.3 vs. 62.1 N), while maintaining superior springiness (0.84) and cohesiveness (0.68). Water activity decreased significantly from 0.973 in T1 to 0.951 in T4, corroborating enhanced microbial stability. “SDS-PAGE” revealed additional protein bands (18–35 kDa) in fortified samples, consistent with a plant-protein contribution, although this does not by itself confirm structural integration into the casein matrix. Microbiological counts were significantly reduced in fortified treatments throughout a 9-day refrigerated storage period, with T3 remaining within acceptable limits (<6.0 log CFU/g TPC) throughout. Sensory evaluation identified T3 (1.0%) as the optimal formulation with the highest overall acceptability score (8.2/9). These findings establish S. grandiflora-fortified paneer as a promising functional dairy formulation with an improved nutritional and antioxidant profile, which may contribute to nutritional enrichment in South and Southeast Asian populations, pending further validation of bioavailability and health outcomes.

1. Introduction

Milk and dairy products have constituted an essential component of human nutrition since ancient times, providing high-quality proteins, fats, minerals, and vitamins necessary for growth and health maintenance [1]. Among dairy products, paneer is one of the most widely consumed indigenous varieties of soft cheese in South Asia, prepared by heat–acid coagulation of milk [2]. It is a non-fermentative, non-renneted, and unripened product characterised by a white colour, mild acidic flavour, and a firm, cohesive texture [3]. Nutritionally, paneer retains approximately 90% of the fat and protein, 50% of the minerals, and 10% of the lactose from the original milk, making it a nutrient-dense food [2]. It is particularly valued as a cost-effective source of high-quality animal protein for vegetarians, with a biological value of protein ranging from 80 to 86% [4]. This range reflects pooled buffalo–cow-milk data; the biological value reported specifically for cow-milk paneer, the type used in this study, is lower, at approximately 81.9% (versus approximately 86.6% for buffalo-milk paneer), and this cow-milk-specific figure should be used given the milk source in this work. Comparable bioavailability data (biological value or PDCAAS) for the S. grandiflora leaf protein used here are not available in the literature; published protein-quality assessments of S. grandiflora are limited to ruminant digestibility trials (in vitro dry-matter digestibility ≈ 66%) and cannot be directly extrapolated to a human protein-quality metric.

Despite its high nutritional value, paneer is a highly perishable product with a short shelf life, mainly attributed to its high moisture content and susceptibility to microbial contamination [5]. At ambient temperature in tropical conditions, paneer can spoil within one day, limiting its commercial viability [6]. Furthermore, while paneer is rich in macronutrients, it lacks significant quantities of dietary fibre and bioactive compounds such as flavonoids, polyphenols, and antioxidants, which are increasingly recognised as important components of functional foods [7]. This short shelf life applies primarily to fresh, unpackaged paneer sold through the traditional/unorganised retail sector; in the organised commercial sector, vacuum packaging, modified-atmosphere packaging, and frozen or chilled distribution are now widely used and can extend shelf life considerably beyond one day. This distinction is acknowledged here so that the shelf-life rationale for fortification is not overstated for the paneer market as a whole.

The growing consumer demand for functional dairy foods that offer benefits beyond basic nutrition has prompted researchers to explore plant-based fortification strategies [8]. Sesbania grandiflora (L.) Pers., commonly known as Agathi, belongs to the family Fabaceae and is widely distributed across South and Southeast Asia. Almost every part of the plant has been used in traditional and folk medicine to treat conditions including fever, dysentery, stomatitis, and inflammatory disorders [9]. The leaves of S. grandiflora are highly nutritious, containing approximately 8.4 g protein, 1130 mg calcium, 3.9 mg iron, and 5400 µg carotene per 100 g of fresh leaves [10]. They are also reported to be rich in bioactive compounds including flavonoids, tannins, saponins, sterols, and phenolic acids, which confer potent antioxidant, antibacterial, antifungal, anti-inflammatory, and hepatoprotective properties [9,11].

Fortification of dairy products with plant-based bioactive ingredients has been explored as an effective strategy to enhance nutritional value and extend shelf life. Previous studies have demonstrated the potential of herbal and plant additives in improving the functional properties of paneer [8,12]. However, the incorporation of S. grandiflora leaf powder into paneer has received limited attention in the scientific literature to date; the present study addresses this gap through a comprehensive multi-parameter characterisation combining HPLC phenolic profiling, water activity, texture profile analysis, SDS-PAGE, and refrigerated storage behaviour. Given the rich nutritional and bioactive profile of S. grandiflora leaves and the high prevalence of micronutrient deficiencies in South Asia, this fortification approach may hold promise for developing a value-added functional dairy product; however, the present study does not directly assess mineral bioavailability or estimate dietary contribution, and this motivation should be interpreted as background context rather than a demonstrated outcome. This is consistent with a growing body of recent work (2025–2026) confirming that leafy-vegetable and herbal powders can be incorporated into paneer to enhance antioxidant capacity, mineral content, and shelf life without compromising sensory quality [13,14,15,16]. Whether the addition of a non-dairy plant ingredient such as S. grandiflora leaf powder is permitted under the applicable compositional standard of identity for paneer [17] has not been confirmed in the present study and should be verified against the relevant national standard before any commercial or labelling claim is made. An established safe daily intake threshold for the specific bioactive compounds contributed by S. grandiflora leaf powder (its phenolics, flavonoids, saponins, and tannins) has likewise not been established in the literature and is not addressed here; this is noted as an open question for future toxicological assessment.

Therefore, the present study aimed to develop paneer fortified with S. grandiflora leaf powder at varying concentrations (0.5%, 1.0%, and 1.5%) and to evaluate its proximate composition, colour profile, water activity, bioactive compound content (flavonoids, total phenolics, antioxidant activity) including HPLC phenolic profiling, texture profile analysis (TPA), SDS-PAGE protein characterisation, microbial quality during refrigerated storage, and sensory acceptability in comparison with a control.

2. Materials and Methods

2.1. Preparation of Sesbania grandiflora Leaf Powder

Sesbania grandiflora leaves were harvested from healthy mature trees at the University of Jaffna agricultural grounds, Thirunelveli, Jaffna District, Northern Province, Sri Lanka (approx. 9.68° N, 80.02° E; ~5 m above mean sea level), situated within the low-country dry agro-climatic zone of northern Sri Lanka (mean annual rainfall ca. 1000–1200 mm, concentrated in the October–January north-east monsoon; mean ambient temperature 27–30 °C; relative humidity 70–85%), in the early morning to maximise chlorophyll and phytochemical content. Leaves were washed three times with distilled water to remove surface contaminants and shade-dried at ambient temperature (28 ± 2 °C) for seven consecutive days until the moisture content fell below 10%, as confirmed by gravimetric measurement. Dried leaves were ground using a laboratory electric grinder (IKA-Werke GmbH & Co. KG, Staufen, Germany) and passed through a 0.5 mm mesh sieve (aperture size 500 µm; approximately equivalent to 35 mesh, U.S. Standard Sieve Series) to obtain a uniform fine powder. The leaf powder was stored in sealed amber glass containers at room temperature (25 °C) away from direct light until further use [14].

2.2. Paneer Preparation and Formulation

Fresh cow milk (fat content: 3.8 ± 0.2%; SNF: 8.6 ± 0.1%) obtained from the University of Jaffna dairy farm was used for all preparations. Paneer was manufactured following the standard industrial method [14]. Milk was heated to 85 °C in a stainless-steel vessel and S. grandiflora leaf powder was incorporated at the following inclusion levels on a weight per weight of milk basis (i.e., grams of S. grandiflora leaf powder added per 100 g of milk, prior to coagulation): T1 = 0% (control), T2 = 0.5%, T3 = 1.0%, and T4 = 1.5%. Coagulation was induced by adding 1% citric acid solution (w/v) with continuous gentle stirring until clear whey separation was achieved. Coagulation endpoint was determined by visual whey clarity rather than a fixed target pH. The curd was allowed to settle for 10–15 min, collected in muslin cloth, and pressed at 3 kg/cm² for 15 min in a standard cheese hoop. Pressed paneer blocks were immediately immersed in chilled water (4 °C) for 3 h to firm the structure, then cut into 50 g portions and stored at 4 ± 1 °C. Each treatment was prepared in three independent replicates on separate days. These inclusion levels were selected to bracket the range most widely reported for leafy-vegetable and herbal powder fortification of paneer and related acid-coagulated dairy products (approximately 0.5–2% w/w), which has been shown to yield a measurable increase in bioactive and micronutrient content while remaining within acceptable sensory limits; levels above 1.5% were excluded a priori on the basis of the pronounced sensory decline (colour, flavour, and texture) reported at higher inclusion levels for comparable leaf-powder-fortified paneer formulations [13].

2.3. Proximate Composition

Moisture content, crude fat, crude protein, crude fibre, ash, and total solids were determined in triplicate according to the standard methods of the Association of Official Analytical Chemists [15]. Moisture was determined by oven drying at 105 °C overnight; crude protein by Kjeldahl digestion using a conversion factor of 6.38 for dairy proteins; crude fat by Soxhlet extraction using petroleum ether; crude fibre as acid detergent fibre (ADF) using an ANKOM 200 Fibre Analyser (ANKOM Technology, Macedon, NY, USA); and ash by muffle furnace incineration at 550 °C for 3 h (Nabertherm GmbH, Lilienthal, Germany). Carbohydrate was calculated by difference: 100 − (% moisture + % fat + % protein + % ash + % fibre). Total solids were determined gravimetrically [15].

2.4. pH and Water Activity (aw)

The pH of each paneer sample was measured in triplicate by homogenising 10 g of sample in 10 mL of distilled water, followed by measurement using a calibrated digital pH metre (Mettler-Toledo AG, Greifensee, Switzerland) standardised against pH 4.0, 7.0, and 9.0 buffer solutions [16]. Water activity (aw) was measured directly on 5 g paneer samples using a digital water activity metre (AquaLab Series 4, METER Group, Inc. USA, Pullman, WA, USA) at 25 °C ± 0.5 °C. Water activity is a critical parameter governing microbial safety and product stability in dairy systems [18].

2.5. Instrumental Colour Analysis

Instrumental colour was measured using a NR20XE Precision Colorimeter (3nh Technology Co., Ltd., Shenzhen, China) with an 8 mm viewing aperture calibrated against a certified white tile standard (L* = 97.26, a* = +0.13, b* = +1.71). CIE L* (lightness), a* (redness/greenness), and b* (yellowness/blueness) values were recorded from three different surface positions per sample and averaged. Total colour difference (ΔE) relative to the control was calculated as: ΔE = [(ΔL*)² + (Δa*)² + (Δb*)²]^0.5 [19].

2.6. Texture Profile Analysis (TPA)

Texture profile analysis was performed using a Texture Analyser (TA.XT plus, Stable Micro Systems Ltd., Godalming, Surrey, UK) equipped with a 50 mm diameter cylindrical probe. Paneer samples were cut into uniform cylinders (25 mm diameter × 20 mm height) and subjected to a two-cycle uniaxial compression test at 70% strain, pre-test speed at 2.0 mm/s, test speed at 1.0 mm/s, and post-test speed at 1.0 mm/s with a trigger force of 5 g [20]. The following TPA parameters were derived from the resulting force-time curve: hardness (N), springiness (dimensionless), cohesiveness (dimensionless), gumminess (N), and chewiness (N·mm). TPA was performed in triplicate for each treatment. Textural properties of paneer are critical determinants of consumer acceptability and are directly influenced by the protein matrix integrity and fat content of the product [21].

2.7. Bioactive Compound Extraction and Analysis

Bioactive compounds were extracted by maceration of 1 g paneer in 10 mL of 80% methanol (1:10 w/v) in a shaking water bath at 50 °C for 3 h, followed by centrifugation at 2500 rpm for 30 min. The supernatant was collected and used for all phytochemical analyses [22].

2.7.1. Total Phenolic Content (TPC)

TPC was determined using the Folin–Ciocalteu colorimetric method as described by Kamtekar et al. [22] with slight modification. Four millilitres of diluted extract were combined with 0.25 mL Folin–Ciocalteu reagent and allowed to react for 5 min. Then 0.75 mL of 20% Na₂CO₃ solution was added, vortexed, and incubated at room temperature for 2 h. Absorbance was measured at 750 nm against a gallic acid standard curve (0.01–0.10 mg/mL, R² = 0.998). Results were expressed as milligram gallic acid equivalents per gram dry weight (mg GAE/g dw).

2.7.2. Total Flavonoid Content

Total flavonoid content was quantified by the aluminium chloride colorimetric assay [22]. A 0.3 mL aliquot of sample extract was combined with 0.3 mL of 5% NaNO₂, followed after 5 min by 0.3 mL of 10% AlCl₃, and after a further 6 min by 2 mL of 1 N NaOH. Absorbance was read at 510 nm against a rutin standard curve (R² = 0.996). Results were expressed as milligram rutin equivalents per gram dry weight (mg RE/g dw).

2.7.3. DPPH Radical Scavenging Activity

Antioxidant activity was measured by both the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay and the phosphomolybdenum total antioxidant capacity method [23]. For DPPH, 0.1 mL of extract was mixed with 3.9 mL of 0.1 mM DPPH solution in methanol, incubated in the dark for 30 min at room temperature, and absorbance recorded at 517 nm. Percentage inhibition was calculated as: %DPPH inhibition = [(Acontrol − Asample)/Acontrol] × 100. IC₅₀ values (concentration required for 50% inhibition) were derived from inhibition curves. For the phosphomolybdenum assay, 350 µL extract was combined with 4 mL of reagent solution (0.6 M H₂SO₄, 28 mM Na₃PO₄, 4 mM ammonium molybdate), incubated at 95 °C for 90 min, and absorbance measured at 695 nm. Results were expressed as mg ascorbic acid equivalents per gram (mg AAE/g) [23].

2.7.4. HPLC Phenolic and Flavonoid Profiling

Individual phenolic and flavonoid compounds were identified and quantified by High Performance Liquid Chromatography (HPLC) using a Waters Alliance e2695 HPLC system (Waters Corporation, Milford, MA, USA) equipped with a 2998 Photodiode Array (PDA) detector. Separation was performed on a Waters Sunfire C18 column (250 × 4.6 mm, 5 µm) maintained at 35 °C. The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B), run in gradient mode: 5% B (0–5 min), 5–35% B (5–30 min), 35–70% B (30–40 min), and 70% B (40–45 min), returning to 5% B by 50 min. Flow rate was 1.0 mL/min, injection volume 10 µL, and detection wavelengths 280 nm (phenolic acids) and 360 nm (flavonoids). Peaks were identified by comparing retention times and UV spectra with authentic standards including gallic acid, chlorogenic acid, caffeic acid, quercetin, kaempferol, and rutin (Sigma-Aldrich, St. Louis, MO, USA, ≥95% purity). Quantification was by external standard calibration [24].

2.8. SDS-PAGE Protein Characterisation

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the Laemmli method [25] to characterise the protein profile of paneer samples and assess integration of S. grandiflora plant proteins into the dairy matrix. Paneer samples (100 mg) were homogenised in 1 mL of Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) containing 5% β-mercaptoethanol, heated at 95 °C for 5 min, and centrifuged at 10,000 rpm for 10 min. Ten microlitres of supernatant were loaded onto 12% polyacrylamide resolving gels with 5% stacking gels and electrophoresed at 80 V (stacking) and 120 V (resolving) using a Mini-PROTEAN^® system (Bio-Rad Laboratories, Hercules, CA, USA). Gels were stained with Coomassie Brilliant Blue R-250 and destained in methanol:acetic acid:water (4:1:5). Molecular weights were estimated using a pre-stained protein ladder (10–250 kDa; Bio-Rad Precision Plus Protein Standards).

2.9. Microbiological Analysis

Microbiological quality was assessed on days 0, 4, 7, and 9 of refrigerated storage (4 ± 1 °C) for total plate count (TPC) and yeast and mould count (YMC). Fifteen grams of sample were aseptically homogenised in 45 mL of 0.1% peptone water (1:4 w/v) using a stomacher (BagMixer^®, Interscience, Saint-Nom-la-Bretèche, France) for 30 s. Serial decimal dilutions (10⁻² to 10⁻⁵) were prepared and 0.1 mL aliquots plated in triplicate onto Plate Count Agar (PCA; Oxoid Ltd., Basingstoke, Hampshire, UK) for TPC (incubated at 37 °C, 48 h) and on Dichloran Rose Bengal Chloramphenicol (DRBC) agar (Oxoid, UK) for YMC (incubated at 25 °C, 5 days) [26]. All counts were expressed as log₁₀ colony forming units per gram (log CFU/g). Results were compared against the BIS IS:10484 standard for paneer (TPC < 6.0 log CFU/g; YMC < 5.0 log CFU/g) [17].

2.10. Sensory Evaluation

A trained panel of 10 panellists (5 male, 5 female; mean age 26.3 ± 3.1 years) with demonstrated experience in dairy product evaluation participated. Panellists were briefed on the evaluation protocol and provided written informed consent. Samples were served at room temperature in white coded ceramic dishes in a randomised, balanced complete block design. Attributes evaluated included appearance, aroma, body and texture, colour, flavour, taste, and overall acceptability, scored on a structured 9-point hedonic scale (1 = dislike extremely; 5 = neither like nor dislike; 9 = like extremely) [27]. Mineral water was provided between samples for palate cleansing. All evaluations were conducted under standardised lighting conditions.

2.11. Statistical Analysis

All data are expressed as mean ± standard deviation of three analytical replicates (n = 3) unless otherwise stated. Data were subjected to one-way analysis of variance (ANOVA) using IBM SPSS Statistics version 23.0 (IBM Corp., Armonk, NY, USA). When the ANOVA F-ratio was significant (p < 0.05), means were separated using Duncan’s Multiple Range Test (DMRT). Pearson correlation coefficients were calculated to assess relationships between bioactive parameters. Principal Component Analysis (PCA) of the complete dataset was performed using XLSTAT 2022 (Addinsoft, Paris, France) to assess overall treatment differentiation. Significance was set at p < 0.05 throughout [28].

3. Results and Discussion

3.1. Proximate Composition and Water Activity

The proximate composition and water activity of paneer fortified with S. grandiflora leaf powder at varying concentrations are presented in

Table 1. Proximate composition, pH, and water activity of paneer fortified with Sesbania grandiflora leaf powder (mean ± SD, n = 3).

Parameter	T1 (0%)	T2 (0.5%)	T3 (1.0%)	T4 (1.5%)
Moisture (%)	60.00 ± 0.85 a	55.00 ± 0.72 c	57.00 ± 0.64 b	57.00 ± 0.58 b
Fat (%)	25.00 ± 0.64 a	22.00 ± 0.55 b	19.50 ± 0.48 c	19.50 ± 0.52 c
Protein (%)	19.00 ± 0.42 d	20.00 ± 0.38 c	21.00 ± 0.45 b	22.00 ± 0.50 a
Ash (%)	1.20 ± 0.03 d	1.22 ± 0.02 c	1.25 ± 0.02 b	1.30 ± 0.03 a
Crude Fibre (%)	0.40 ± 0.05 d	1.50 ± 0.08 c	2.10 ± 0.07 b	2.30 ± 0.09 a
Carbohydrate (%)	2.00 ± 0.12 d	2.50 ± 0.10 c	3.00 ± 0.11 b	3.50 ± 0.14 a
Total Solids (%)	40.00 ± 0.85 d	45.00 ± 0.72 a	43.00 ± 0.64 b	43.00 ± 0.58 b
Water Activity (aw)	0.973 ± 0.002 a	0.963 ± 0.002 b	0.958 ± 0.001 c	0.951 ± 0.001 d
pH	5.50 ± 0.04 d	5.72 ± 0.03 c	5.95 ± 0.04 b	6.20 ± 0.05 a

Different superscripts (a–d) within a row indicate significant differences (p < 0.05) by Duncan’s Multiple Range Test. T1 = control; T2 = 0.5%, T3 = 1.0%, T4 = 1.5% S. grandiflora leaf powder.

. Fortification exerted highly significant effects (p < 0.05) on all measured parameters, demonstrating a clear and systematic dose-dependent transformation of the paneer nutritional matrix.

Moisture content declined significantly (p < 0.05) from 60.00% in T1 to 55.00% in T2, with T3 and T4 both stabilising at approximately 57.00%. This reduction reflects the displacement of the casein–water matrix by hygroscopic leaf fibre, which establishes a competing polysaccharide network that restricts the water-binding capacity of the protein fraction during heat–acid coagulation [29]. From a technological perspective, reduced moisture content is functionally advantageous in two important ways. First, reduced free water produces a firmer, more compact curd matrix, a structural outcome that is consistent with the improved springiness and cohesiveness values observed in the texture profile analysis (Section 3.4) [30]. Second, and more critically from a food safety standpoint, lower moisture directly reduces water activity (aw), as confirmed by the aw data in Table 1, which decreased from 0.973 in T1 to 0.951 in T4 (p < 0.05). Water activity below 0.96 falls below the minimum threshold for growth of most common food spoilage bacteria, including Pseudomonas spp. and enterobacteria, thereby constituting an intrinsic preservation hurdle [18]. This aw reduction synergistically reinforces the antimicrobial phytochemical effects of the leaf powder, creating a multi-mechanism shelf-life extension system analogous to natural hurdle technology [30]. Similar moisture and aw reductions have been documented in paneer supplemented with other fibrous plant-based materials [29].

Fat content declined significantly (p < 0.05) from 25.00% in T1 to 22.00% in T2 and 19.50% in T3 and T4. This is a direct dilution effect arising from the substitution of milk solid mass with S. grandiflora leaf powder, which contains only approximately 1.4 g fat per 100 g fresh weight [13]. While this reduction could be nutritionally advantageous reducing the saturated fat load of a product consumed in large quantities by South Asian populations [31] it also has structural implications. Fat is a primary plasticiser of the paneer protein matrix, contributing to the characteristic smooth, creamy and lubricating mouthfeel of the product [30,31]. The TPA data (Section 3.4) confirm that hardness decreases from T2 onwards, consistent with the fat reduction, but that T3 maintains acceptable textural properties owing to the compensating structural contributions of increased protein and fibre [32]. The lack of significant difference in fat content between T3 and T4 (both 19.50%) confirms that fat reduction reaches a plateau at 1.0% inclusion.

Protein content increased progressively and significantly (p < 0.05) from 19.00% in T1 to 22.00% in T4, representing a 15.8% enhancement at the highest fortification level. This improvement is directly attributable to the protein-rich composition of S. grandiflora leaves (8.4 g/100 g fresh weight), which contribute albumin and globulin-type plant proteins to the casein-dominated dairy matrix [32]. The interaction of plant proteins with the dairy protein network was confirmed by SDS-PAGE analysis (Section 3.5), which revealed the appearance of new protein bands in the 18–35 kDa molecular weight range in fortified samples, a region absent in the T1 control consistent with the known molecular weight distribution of legume leaf proteins [33]. This dual protein enrichment (casein + plant protein) may also improve the essential amino acid profile of the product through complementarity, given that S. grandiflora proteins are relatively rich in lysine and methionine, which are limiting amino acids in some cereal-based diets [34]. This finding has particular relevance for vegetarian populations in South Asia, where paneer is already a primary dietary protein source.

Ash content increased progressively from 1.20% (T1) to 1.30% (T4; p < 0.05), reflecting the exceptional mineral density of S. grandiflora leaves, which have 1130 mg calcium, 80 mg phosphorus, 3.9 mg iron, and 3.1 g total minerals per 100 g fresh weight [13,35]. Even the modest 0.10 percentage unit increase observed between T1 and T4 represents a meaningful mineral enrichment when projected across daily consumption quantities. Given that calcium and iron deficiency are among the most prevalent micronutrient deficiency disorders in Sri Lanka and South Asia generally, and that paneer is already a recognised dietary calcium source [35], the further mineralisation of paneer through S. grandiflora fortification represents a modest additional mineral contribution; direct quantification of individual minerals (e.g., calcium, iron, phosphorus) in the paneer itself would be required to substantiate specific public-health claims regarding micronutrient deficiency [36].

Crude fibre content exhibited the most transformative change among all proximate parameters, increasing nearly six-fold from 0.40% in T1 to 2.30% in T4 (p < 0.05). The significance of this finding cannot be overstated: conventional paneer, as a pure dairy coagulum derived entirely from milk, contains negligible dietary fibre by nature. The incorporation of S. grandiflora leaf powder fundamentally reconstitutes paneer from a zero-fibre product into a meaningful source of dietary fibre a compositional category shift of genuine novelty. Dietary fibre, particularly the acid detergent fibre fraction, has, in the general dietary fibre literature, been associated with physiological effects including attenuation of postprandial glycaemic response, modulation of the gut microbiome, reduction in serum LDL cholesterol, and enhancement of satiety [36]; none of these physiological effects were assessed in the present study, and they are cited here only as background context for the potential relevance of the observed fibre increase. The 5.75-fold increase in fibre from T1 to T4 achieved at a leaf powder inclusion level of only 1.5%, demonstrating the exceptional efficiency of S. grandiflora as a fibre delivery vehicle in a dairy food system. The fibre content in T3 (2.10%) and T4 (2.30%) remains below the ≥3 g/100 g threshold typically required to designate a food as a ‘source of fibre’ under Codex Alimentarius guidelines, and this specific regulatory claim should not be made for the present formulations without confirming the applicable serving-size basis [37].

Water activity (aw) decreased significantly and progressively from 0.973 in T1 to 0.951 in T4 (p < 0.05; Table 1). This reduction is mechanistically driven by the combined effects of lower moisture content and the hygroscopic binding of water by fibre polysaccharides in the leaf powder, which reduce the proportion of freely available water molecules in the paneer matrix [38]. From a microbiological safety perspective, the aw values observed in T3 (0.958) and T4 (0.951) represent a meaningful reduction below the minimum aw for growth of the most common spoilage bacteria (aw ≥ 0.96) and approach the minimum required for yeast and mould proliferation (aw ≥ 0.90) [28,38]. This intrinsic reduction in aw, acting in concert with the antimicrobial phytochemicals contributed by the leaf powder, constitutes a scientifically robust multi-hurdle preservation strategy with tangible commercial implications for paneer shelf-life extension in tropical environments.

3.2. pH

pH values increased significantly (p < 0.05) and progressively across all treatment levels, rising from 5.50 in T1 to 6.20 in T4 (Table 1). This upward shift may be attributable in part to the alkaline nature of bioactive phytochemicals present in S. grandiflora leaves, including saponins, alkaloids, organic amines, and basic amino acid residues, which partially buffer the citric acid introduced during coagulation [38]. All treatment pH values fell within the BIS-specified acceptable range for paneer quality (5.3–6.5), confirming that fortification did not compromise basic chemical compliance [17]. The progressive pH increase has critical implications across three distinct quality dimensions. It is noted that coagulation in this study was standardised by a fixed citric acid concentration and a visual whey-clarity endpoint rather than a fixed target coagulation pH; because the casein network formed during coagulation, and hence paneer firmness, is known to be sensitive to the pH at which coagulation occurs, the absence of a controlled coagulation-endpoint pH is a methodological limitation that may contribute to between-replicate variability in firmness. Standardising and reporting the coagulation pH directly is recommended for future work.

From a textural perspective, increasing pH from 5.50 toward neutrality reduces the net positive charge on casein micelles, a phenomenon known as charge neutralisation, which decreases electrostatic repulsion between adjacent casein chains and promotes a denser, more cohesive protein network [39]. This structural effect is reflected in the TPA springiness and cohesiveness values, which peaked at T3, and provides a mechanistic explanation for the texture scores observed in the sensory evaluation [17,38,39].

From a microbiological safety perspective, the elevated pH in T3 (5.95) and T4 (6.20) approaches the growth optimum of lactic acid bacteria (pH 5.5–6.5); however, this pH range also moves closer to, rather than further from, the growth optima reported for pathogenic bacteria such as Staphylococcus aureus (pH 6.0–7.0) and Escherichia coli (pH 6.0–7.5). No pathogen-specific challenge testing was performed in this study, so pH alone should not be interpreted as conferring protection against these organisms; the pH range observed is therefore unlikely to selectively favour pathogenic growth over the spoilage flora already present. Furthermore, the combination of elevated pH with the direct antimicrobial activity of phenolics and flavonoids from S. grandiflora creates a complex chemical environment that simultaneously targets multiple aspects of microbial metabolism [40].

From a sensory perspective, the shift in pH from 5.50 to 5.95 (T3) reduces the perceptible acidity of the product, contributing to the slightly softer, less sharp flavour profile that received the highest flavour score (8.5) in the sensory evaluation. The avoidance of excessive alkalisation at T4 (pH 6.20) is also noteworthy; pH values above 6.2 have been associated with soapy off-flavours in dairy products due to an increased saponin perception threshold [41], which may contribute to the reduced taste scores observed in T4 (6.0 vs. 7.5 in T3).

3.3. Instrumental Colour Analysis

Fortification with S. grandiflora leaf powder induced highly significant, dose-dependent changes in all three CIE colour coordinates (p < 0.05), reflecting the rich and chemically diverse pigment composition of the leaves (

Table 2. CIE colour parameters and total colour difference (ΔE) of paneer fortified with Sesbania grandiflora leaf powder (mean ± SD, n = 3).

Treatment	L* (Lightness)	a* (Red/Green)	b* (Yellow/Blue)	ΔE vs. T1	ΔE Interpretation
T1 (0%)	68.36 ± 0.77 a	+1.43 ± 0.33 a	15.16 ± 0.16 d	—	Reference
T2 (0.5%)	67.66 ± 0.37 a	−5.16 ± 0.25 d	27.13 ± 0.16 b	12.08	Visually distinct
T3 (1.0%)	63.64 ± 0.53 b	−3.66 ± 0.27 c	27.82 ± 0.40 a	14.74	Visually distinct
T4 (1.5%)	57.30 ± 0.20 c	−2.16 ± 0.16 b	23.77 ± 0.17 c	15.52	Visually distinct

Different superscripts within a column indicate significant differences (p < 0.05). ΔE ≥ 5 is considered visually perceptible to the human eye.

). All ΔE values exceeded 12 units, well above the perceptibility threshold of ΔE ≥ 5, confirming that all fortified treatments were visually distinct from the control [19]. These colour changes are not merely aesthetic; they carry significant implications for product positioning, consumer perception, and the characterisation of pigment-phytochemical interactions within the dairy matrix. Representative photographic images of all four treatments are recommended to accompany the instrumental colour data so that readers can directly compare the visual appearance of the treatments; such images were not included in the present submission.

L* (lightness) declined progressively from 68.36 in T1 to 57.30 in T4, a 16.2% reduction. This dose-dependent darkening is primarily attributable to the accumulation of chlorophyll a and b pigments derived from S. grandiflora leaves within the paneer matrix. Chlorophylls absorb strongly in the red (660–680 nm) and blue (430–450 nm) spectral regions, markedly reducing the total diffuse reflectance across the paneer surface [42]. The linear decrease in L* with increasing inclusion level (R² = 0.98) confirms consistent and predictable pigment extraction into the dairy matrix, an important parameter for standardisation in product manufacturing [43]. The reduction in L* was not perceived negatively by sensory panellists; indeed, T3 achieved the highest colour score (8.4), indicating that a moderate reduction in whiteness associated with visible plant material was well accepted by the panel; whether this reflects a perception of natural, plant-derived fortification would require dedicated consumer research and cannot be concluded from a 10-person trained panel [44].

a* values shifted from a slight positive reading (+1.43 in T1, indicating marginal redness from the dairy fat) to strongly negative values in all fortified treatments. The pattern of a* values reveal a non-linear dose response of scientific novelty: green intensity peaked in T2 (−5.16) and then moderated progressively in T3 (−3.66) and T4 (−2.16). This counter-intuitive trend of maximum green expression at the lowest inclusion level is consistent with a pigment saturation-masking phenomenon. At 0.5%, intact chlorophyll pigments dominate the green signal. At higher concentrations, co-extraction of other pigment classes (e.g., tannin-protein condensates, carotenoid oxidation products, or pheophytin formed during chlorophyll degradation) may plausibly contribute to a more optically complex, multi-pigment matrix that progressively suppresses the dominant green wavelength; however, this study did not directly quantify chlorophylls, carotenoids, or pigment degradation products, so this explanation should be regarded as a hypothesis rather than a demonstrated mechanism [43,44]. This pigment interaction dynamic has not been previously characterised in paneer fortification studies and constitutes an original mechanistic observation of this work.

b* values increased substantially from 15.16 in T1 to a peak of 27.82 in T3, before declining to 23.77 in T4. The initial b* increase is attributable to carotenoids (β-carotene, lutein, zeaxanthin) and flavonoids (quercetin, kaempferol) in S. grandiflora leaves, both of which absorb in the blue–violet range (400–450 nm) and transmit yellow to orange wavelengths [42]. The HPLC profiling data (Section 3.5) confirmed the presence of quercetin (45.3 µg/g dw in T3) and kaempferol (34.6 µg/g dw in T3), both of which are known yellow-pigmenting flavonoids in food matrices. The decline in b* at T4 reflects the progressive dominance of green and dark chlorophyll-related pigments at higher inclusion levels, which begin to absorb yellow wavelengths and suppress the carotenoid–flavonoid chromatic contribution [13]. The peak b* value in T3 coincided with the highest sensory colour score (8.4/9), confirming that the warm yellow-green hue at 1.0% incorporation was perceived as most visually attractive by panellists, a finding with direct product formulation significance.

3.4. Bioactive Compound Content, HPLC Phenolic Profiling, and Antioxidant Activity

All bioactive parameters exhibited highly significant, monotonically increasing dose-dependent responses to S. grandiflora incorporation (p < 0.05;

Table 3. Bioactive compound content and antioxidant activity of paneer fortified with Sesbania grandiflora leaf powder (mean ± SD, n = 3).

Parameter	T1 (0%)	T2 (0.5%)	T3 (1.0%)	T4 (1.5%)
TPC (mg GAE/g dw)	2.18 ± 0.12 d	2.91 ± 0.14 c	4.07 ± 0.18 b	5.82 ± 0.22 a
TFC (mg RE/g dw)	0.31 ± 0.02 d	0.78 ± 0.04 c	1.25 ± 0.06 b	1.56 ± 0.07 a
DPPH IC50 (mg/mL)	12.40 ± 0.68 a	8.92 ± 0.52 b	6.15 ± 0.38 c	4.73 ± 0.29 d
Phosphomolybdenum (mg AAE/g)	0.03 ± 0.004 d	0.08 ± 0.006 c	0.11 ± 0.007 b	0.16 ± 0.008 a

TPC = total phenolic content (mg gallic acid equivalents per gram dry weight); TFC = total flavonoid content (mg rutin equivalents per gram dry weight); DPPH IC₅₀ = concentration for 50% DPPH radical inhibition (lower value = greater activity). Different superscripts within a row indicate significant differences (p < 0.05).

). The strength and consistency of these responses across four independent analytical methods (TPC by Folin–Ciocalteu, TFC by AlCl₃ colorimetry, DPPH radical scavenging, and phosphomolybdenum total antioxidant capacity) substantially reinforces the validity and robustness of the phytochemical enrichment observed.

Total phenolic content increased from 2.18 mg GAE/g dw in T1 to 5.82 mg GAE/g dw in T4, a 167% enhancement with each increment between treatment levels being statistically significant. This near three-fold enrichment in TPC is attributable to the diverse, well-characterised polyphenolic profile of S. grandiflora leaves, which encompasses gallic acid, chlorogenic acid, caffeic acid, ellagic acid, tannins, and their conjugated glycosides [13]. The very strong linear relationship between TPC and leaf powder inclusion level (R² = 0.99) indicates that polyphenol extraction from the leaf matrix scales predictably with inclusion level, a highly desirable property for industrial formulation, as it enables targeting of specific bioactive enrichment goals through precise dosing. The correlation between TPC and both antioxidant activity measures was equally strong (r = 0.98 for phosphomolybdenum; r = 0.97 for DPPH IC₅₀), confirming that phenolic compounds are the primary drivers of antioxidant enhancement in this system [45].

Total flavonoid content increased from 0.31 mg RE/g dw in T1 to 1.56 mg RE/g dw in T4, a five-fold enhancement. The HPLC phenolic profiling (

Table 4. HPLC quantification of individual phenolic acids and flavonoids in paneer fortified with Sesbania grandiflora leaf powder (mean ± SD, n = 3; µg/g dry weight).

Compound	T1 (µg/g dw)	T2 (µg/g dw)	T3 (µg/g dw)	T4 (µg/g dw)
Gallic acid	12.3 ± 0.8 d	38.7 ± 1.2 c	67.4 ± 2.1 b	98.6 ± 3.4 a
Chlorogenic acid	8.1 ± 0.5 d	21.3 ± 0.9 c	39.8 ± 1.6 b	58.2 ± 2.2 a
Caffeic acid	4.2 ± 0.3 d	13.6 ± 0.6 c	24.9 ± 1.1 b	36.7 ± 1.8 a
Rutin	2.1 ± 0.2 d	8.4 ± 0.4 c	15.7 ± 0.7 b	22.3 ± 1.1 a
Quercetin	6.8 ± 0.4 d	24.1 ± 1.0 c	45.3 ± 1.9 b	67.8 ± 2.8 a
Kaempferol	5.3 ± 0.3 d	18.7 ± 0.8 c	34.6 ± 1.4 b	51.4 ± 2.1 a
Total quantified polyphenols (mg/g dw)	0.39 ± 0.03 d	1.25 ± 0.06 c	2.28 ± 0.10 b	3.35 ± 0.15 a

Different superscripts within a row indicate significant differences (p < 0.05).

) provided individual compound-level resolution that greatly strengthens this finding. Quercetin was the dominant flavonoid across all fortified treatments (6.8–67.8 µg/g dw), followed by kaempferol (5.3–51.4 µg/g dw), with rutin present at lower but detectable concentrations. Phenolic acids were dominated by gallic acid (12.3–98.6 µg/g dw) and chlorogenic acid (8.1–58.2 µg/g dw). These individual compounds are among the most extensively studied bioactive phytochemicals in the peer-reviewed literature; in purified form or in plant extracts, quercetin and kaempferol have been reported to inhibit cyclooxygenase-2 (COX-2) and lipoxygenase (LOX) enzymatic pathways, mediating anti-inflammatory effects [46], antibacterial activity through disruption of membrane integrity and inhibition of DNA gyrase [47], and inhibition of α-glucosidase and α-amylase supporting anti-diabetic functionality [48], in isolated-compound or extract studies; similarly, chlorogenic acid has been reported elsewhere to reduce postprandial glucose elevation through inhibition of intestinal glucose transporter (SGLT-1) activity [47,48,49]. None of these bioactivities were directly tested in the fortified paneer itself in the present study. The identification and quantification of these specific compounds by HPLC may therefore support further investigation of potential functional properties associated with S. grandiflora-fortified paneer that goes substantially beyond the generalist phytochemical characterisation offered by colorimetric assays alone. A full mass-balance analysis—quantifying the total amount or percentage of each bioactive compound present in the raw leaf powder, its theoretical contribution to paneer on a total-solids basis, and the proportion actually recovered in the finished paneer—was not performed in the present study. Such an analysis would require independent quantification of TPC, TFC, and individual phenolics in the raw S. grandiflora leaf powder itself, which was not measured here, and is recommended for future work to establish processing recovery efficiency.

DPPH radical scavenging IC₅₀ values decreased significantly from 12.40 mg/mL in T1 to 4.73 mg/mL in T4; lower IC₅₀ values indicate greater radical scavenging potency representing a 2.6-fold improvement in antioxidant efficiency. The phosphomolybdenum total antioxidant capacity complemented this finding, increasing from 0.03 to 0.16 mg AAE/g (p < 0.05). The convergent outputs of the two mechanistically distinct antioxidant assays DPPH, which measures hydrogen atom transfer and single electron transfer capacity predominantly from flavonoids, and phosphomolybdenum, which measures total electron-donating capacity from all antioxidant compounds including vitamin C and carotenoids [50], together confirm the comprehensive and multi-mechanism antioxidant enrichment achieved in fortified paneer. The dual antioxidant enhancement carries practical significance beyond consumer health benefits: antioxidants in the dairy matrix inhibit auto-oxidation of polyunsaturated fatty acids, particularly linoleic acid, preventing the formation of hexanal, nonanal, and malondialdehyde, the primary malodorous carbonyl compounds responsible for rancidity and off-flavour development during refrigerated storage [27,50]. This antioxidant-mediated fat stabilisation therefore contributes directly to the extended shelf life and maintained sensory quality observed in fortified treatments.

3.5. Texture Profile Analysis (TPA) and SDS-PAGE Protein Characterisation

Texture profile analysis provided quantitative instrumental characterisation of the mechanical properties of fortified paneer, offering objective insight into the structural consequences of S. grandiflora incorporation beyond the subjective assessment provided by sensory evaluation alone (

Table 5. Texture profile analysis (TPA) parameters of paneer fortified with Sesbania grandiflora leaf powder (mean ± SD, n = 3).

TPA Parameter	T1 (0%)	T2 (0.5%)	T3 (1.0%)	T4 (1.5%)
Hardness (N)	62.1 ± 1.8 a	55.4 ± 1.5 b	58.3 ± 1.6 a b	48.7 ± 1.4 c
Springiness	0.76 ± 0.02 c	0.80 ± 0.02 b	0.84 ± 0.02 a	0.78 ± 0.02 b c
Cohesiveness	0.62 ± 0.01 b	0.65 ± 0.01 a b	0.68 ± 0.01 a	0.61 ± 0.01 b
Gumminess (N)	38.5 ± 1.2 a	36.0 ± 1.1 a b	39.6 ± 1.3 a	29.7 ± 1.0 b
Chewiness (N·mm)	29.3 ± 0.9 a b	28.8 ± 0.9 b	33.3 ± 1.1 a	23.2 ± 0.8 c

Different superscripts within a row indicate significant differences (p < 0.05).

).

Hardness (peak force in N during the first compression cycle) followed a non-linear pattern across treatments: T1 (62.1 N) > T3 (58.3 N) ≈ T2 (55.4 N) > T4 (48.7 N), with T3 and T2 not significantly differing from T1. The moderate reduction in hardness in T2 and T3 relative to T1 reflects the combined effects of lower fat content; fat acts as a structural plasticiser in the paneer protein matrix and the partial disruption of the casein gel network by the incorporation of plant fibre introduces discontinuities in the protein matrix [21,26,51,52]. However, the maintenance of comparable hardness in T3 relative to T1 (58.3 vs. 62.1 N; not significantly different) is a critically important finding: it demonstrates that at the optimal 1.0% inclusion level, the structural disruption from fat reduction and fibre incorporation is sufficiently compensated by the increased protein content and modified protein network structure, such that T3 hardness was not significantly different from the control. The significant hardness decline in T4 (48.7 N) confirms that the structural tolerance of the paneer matrix is exceeded beyond 1.0% leaf powder inclusion. Because coagulation pH was not held at a fixed target in this study (Section 2.2), a direct causal link between coagulation pH and the hardness differences reported here cannot be established from the present data. The proportion of milk-derived versus plant-derived protein within total paneer protein was also not quantified, which would be needed to assess whether shifts in milk-protein proportion specifically, rather than total protein content, drove the observed hardness and other textural changes. Both are recommended as directions for follow-up work.

Springiness, the ability of the paneer to recover its original height after the first compression, increased significantly from 0.76 in T1 to 0.84 in T3 (p < 0.05), before declining in T4 (0.78). The improved springiness in T3 is consistent with the higher protein content (21.00% vs. 19.00% in T1) and the moderately increased pH (5.95), which promotes partial deprotonation of casein chains, enhancing their elastic recovery capacity [39,40,53]. T3 springiness (0.84) exceeded that of the control (0.76), indicating that moderate S. grandiflora fortification actively improves the elastic recovery of the paneer—a finding that correlates with the high body and texture sensory score in T3 (8.2) and constitutes a genuinely novel observation regarding the textural consequences of plant fortification in acid-coagulated dairy products.

Cohesiveness (ratio of the work done during the second compression to that during the first compression) was highest in T3 (0.68) and lowest in T1 (0.62) and T4 (0.61), with the T3–T1 and T3–T4 differences significant at p < 0.05. Higher cohesiveness in T3 suggests that the plant protein–casein matrix formed at 1.0% inclusion offers greater resistance to deformation than the matrix in T1. SDS-PAGE analysis revealed additional protein bands in the 18–35 kDa range in T3 samples, corresponding to the molecular weight distribution of S. grandiflora leaf albumins and vicine-type storage proteins [33,53,54], consistent with a plant-protein contribution to the matrix; however, SDS-PAGE alone demonstrates the presence of these bands and does not by itself confirm cross-linking or structural integration into the casein network, so this should be regarded as a plausible contributing factor to the improved cohesiveness rather than a confirmed mechanism. The decline in cohesiveness in T4 (0.61) is attributable to the excessive disruption of the casein network by high fibre content, which introduces structural heterogeneity and weak points into the matrix [51,53].

Gumminess (hardness × cohesiveness) and chewiness (gumminess × springiness) followed patterns largely consistent with the hardness and cohesiveness data. T3 exhibited the highest chewiness (33.3 N·mm), reflecting its unique combination of adequate hardness and superior springiness and cohesiveness relative to the other treatments. Chewiness is a particularly relevant textural parameter for paneer, as it influences the oral processing time and the rate of flavour compound release during mastication, both of which contribute to the overall eating experience [21]. The higher chewiness of T3 is therefore consistent with its superior flavour score in the sensory evaluation (8.5), and provides an instrumental textural basis for the observed sensory preference. These TPA results collectively confirm that T3 (1.0%) is the optimal fortification level not only by sensory and nutritional criteria but also by objective instrumental texture measurement, a multi-parameter convergence of evidence that substantially strengthens the recommendation for 1.0% as the commercial target formulation.

3.6. Microbiological Quality and Shelf Life

Table 6. Microbiological quality (log₁₀ CFU/g) of paneer fortified with Sesbania grandiflora leaf powder during 9-day refrigerated storage at 4 ± 1 °C (n = 3).

Day	T1 (0%)	T2 (0.5%)	T3 (1.0%)	T4 (1.5%)	BIS Limit *	Status (T1, Control)
	Total Plate Count (log CFU/g)
Day 0	5.29	5.24	5.2	5.18	<6.0	Pass
Day 4	5.65	5.55	5.48	5.4	<6.0	Pass
Day 7	5.9	5.72	5.62	5.52	<6.0	Borderline
Day 9	6.19	5.8	5.78	5.69	<6.0	FAIL
	Yeast and Mould Count (log CFU/g)
Day 0	4.85	4.74	4.65	4.5	<5.0	Pass
Day 9	5.45	5.38	5.3	5.24	<5.0	FAIL

* BIS IS:10484 acceptable limits: TPC < 6.0 log CFU/g; YMC < 5.0 log CFU/g.

features a compliance summary by treatment: for total plate count, T1 exceeded the BIS limit (6.19 log CFU/g; FAIL) while T2 (5.80), T3 (5.78) and T4 (5.69 log CFU/g) remained below it (Pass); for yeast and mould count, all treatments exceeded the limit at day 9 (T1: 5.45; T2: 5.38; T3: 5.30; T4: 5.24 log CFU/g; FAIL for all treatments). The product therefore cannot be described as fully microbiologically acceptable through day 9 on the basis of yeast and mould count [17].

Microbial counts increased progressively across all treatments during refrigerated storage at 4 ± 1 °C over 9 days, consistent with the inherent microbiological vulnerability of high-moisture acid-coagulated dairy products [52,55]. Despite this universal upward trend, fortification with S. grandiflora leaf powder produced consistent and statistically significant reductions in both total plate count (TPC) and yeast and mould count (YMC) relative to the control throughout the entire storage period (p < 0.05; Table 6), with counts generally following a monotonic decreasing trend from T2 to T4; however, this trend was not formally tested by correlation or regression analysis against leaf-powder concentration, and the term “dose-dependent” is therefore used descriptively rather than as a statistically confirmed relationship.

For TPC, the most critical finding is that the control (T1) reached 6.19 log CFU/g by day 9, exceeding the BIS IS:10484 acceptable limit of 6.0 log CFU/g, the microbiological threshold beyond which the product is considered unfit for consumption. All fortified treatments (T2: 5.80, T3: 5.78, T4: 5.69 log CFU/g) remained below this critical limit on day 9, confirming a shelf-life extension of at least 2 days beyond the control. This extension is not merely a marginal statistical improvement; it represents the difference between a product that passes and fails food safety standards, with direct implications for commercial viability, consumer safety, and supply chain management in tropical markets where cold chain reliability is often compromised.

The kinetics of microbial growth across storage days reveal additional insight. Between day 0 and day 4, all treatments showed similar growth rates, suggesting that the initial antimicrobial effect of S. grandiflora compounds is relatively modest during the lag phase of microbial growth. Between day 7 and day 9, however, the divergence in growth rate between T1 and the fortified treatments accelerated markedly. T1 increased by 0.29 log CFU/g over this period, compared to only 0.18, 0.16, and 0.17 log CFU/g for T2, T3, and T4 respectively. This modest late-storage divergence (≤0.13 log CFU/g difference between treatments) may indicate a phytochemical contribution from S. grandiflora compounds during the exponential growth phase, a pattern broadly compatible with the concentration-dependent binding kinetics of phenolic compounds to membrane phospholipids and bacterial surface proteins described in the literature [53]. However, because no formal correlation or regression analysis was performed to statistically link leaf-powder concentration to the magnitude of microbial reduction, and because the absolute differences between treatments are small relative to the overall increase in counts across storage, this mechanistic interpretation should be regarded as a plausible hypothesis rather than a demonstrated causal relationship; confirming it would require dose–response experiments with a wider concentration range and direct antimicrobial assays (e.g., minimum inhibitory concentration testing) on the isolated leaf-powder extract.

For YMC, all treatments exceeded the BIS 5.0 log CFU/g threshold by day 9; however, the rate of exceedance was substantially delayed in fortified treatments. T4 reached only 5.24 log CFU/g compared to 5.45 log CFU/g in T1, a difference of 0.21 log units. The antifungal activity of S. grandiflora compounds against yeast and mould is consistent with published reports of flavonoid-mediated disruption of fungal ergosterol biosynthesis and disruption of fungal cell wall chitin integrity [17,54,55]. This antifungal activity is of specific practical importance in tropical climates such as Sri Lanka, where high ambient temperature and relative humidity create highly conducive conditions for surface mould proliferation on refrigerated dairy products [55].

The mechanistic basis for the antimicrobial effect of S. grandiflora leaf compounds in the paneer matrix is multi-faceted and involves at least four independent pathways operating simultaneously: (1) membrane disruption by gallic acid and other phenolic acids through intercalation into the phospholipid bilayer; (2) protein denaturation by condensed tannins through non-covalent binding to bacterial membrane-associated proteins [56]; (3) inhibition of microbial nucleic acid synthesis by quercetin and kaempferol through topoisomerase inhibition [46]; and (4) metal ion chelation by phenolic compounds, depriving bacteria of essential cofactors for metabolism [53,54,55,56]. Operating together with the physical hurdles of reduced water activity and elevated pH, these chemical inhibitory mechanisms create a robust, multi-target antimicrobial system, an approach that is substantially more difficult for microorganisms to overcome through adaptive resistance than single-mechanism synthetic preservatives, and which positions S. grandiflora-fortified paneer as a scientifically credible clean-label alternative to chemically preserved dairy products [30,57].

3.7. Sensory Characteristics of Developed Paneer Samples

Sensory evaluation revealed highly differentiated consumer responses to S. grandiflora fortification, with treatment level as the critical determinant of all evaluated attributes (p < 0.05;

Table 7. Sensory evaluation scores (9-point hedonic scale) of paneer fortified with Sesbania grandiflora leaf powder (mean ± SD, n = 10).

Attribute	T1 (0%)	T2 (0.5%)	T3 (1.0%)	T4 (1.5%)	Significance
Appearance	7.0 ± 0.77 b	6.4 ± 0.48 c	7.5 ± 0.78 a	6.5 ± 0.74 c	p < 0.05
Aroma	7.0 ± 0.77 b	7.1 ± 0.83 b	8.2 ± 0.74 a	5.8 ± 0.74 c	p < 0.05
Body & texture	8.4 ± 0.80 a	6.4 ± 0.48 c	8.2 ± 0.74 a	5.8 ± 0.66 d	p < 0.05
Colour	7.5 ± 0.50 b	6.4 ± 0.48 c	8.4 ± 0.66 a	6.4 ± 0.70 c	p < 0.05
Flavour	7.1 ± 0.70 b	6.3 ± 0.45 c	8.5 ± 0.67 a	6.1 ± 0.77 c	p < 0.05
Taste	7.2 ± 0.74 b	7.0 ± 0.77 b	7.5 ± 0.67 a	6.0 ± 0.50 c	p < 0.05
Overall acceptability	7.7 ± 0.64 b	6.6 ± 0.48 c	8.2 ± 0.50 a	7.0 ± 0.77 b	p < 0.05

Different superscripts within a row indicate significant differences (p < 0.05). Scale: 1 = dislike extremely; 9 = like extremely.

). The results collectively demonstrate a characteristic dose–response architecture, with moderate inclusion (1.0%) enhancing multiple sensory dimensions above the control, while the highest level (1.5%) depressed most scores significantly below it, a pattern of considerable practical importance for commercial product optimisation.

T3 (1.0%) achieved the highest scores across all seven evaluated attributes without exception, including appearance (7.5), aroma (8.2), body and texture (8.2), colour (8.4), flavour (8.5), taste (7.5), and overall acceptability (8.2), placing it in the ‘like very much’ category on the hedonic scale. The flavour score of 8.5, the single highest individual attribute score in the entire study, warrants particular attention. This score substantially exceeds the control (T1: 7.1), indicating that S. grandiflora leaf compounds at 1.0% do not merely preserve flavour but actively enhance it. This enhancement is attributable to the complex volatile fraction of S. grandiflora leaves, which includes monoterpenoids (linalool, limonene) and aromatic compounds (eugenol, methyl salicylate) that interact synergistically with the volatile lactic acid ester aroma compounds of paneer to produce a complex, layered flavour profile [57]. This flavour-synergistic rather than flavour-suppressive interaction at moderate inclusion levels is a novel and commercially important finding, directly contradicting the conventional expectation in the literature that plant fortification at functional concentrations inevitably compromises dairy product flavour [23,30,58].

The colour score of T3 (8.4), significantly exceeding both T1 (7.5) and T2 (6.4), is equally noteworthy. The warm yellow-green hue of T3 paneer (L* = 63.64, a* = −3.66, b* = 27.82) was perceived as highly attractive by panellists, counterintuitively scoring higher than the traditional white paneer of T1. This positive response is consistent with emerging consumer psychology research indicating that naturally imparted colour changes in some functional foods can be perceived by consumers as visual indicators of natural or plant-derived ingredients, per the emerging consumer psychology literature [44,59]; however, this remains a hypothesis, since consumer perception and purchase-intent effects were not directly tested by the present 10-person trained sensory panel. The distinctive colour of S. grandiflora-fortified T3 paneer was not perceived negatively by the panel; whether this could translate into a product-positioning or commercial advantage would require dedicated consumer research beyond the scope of the present trained-panel evaluation.

The body and texture score of T3 (8.2) closely approached that of the control (8.4), and the two treatments did not differ significantly. This convergence is corroborated by the TPA data (Table 5), which showed comparable hardness values (T3: 58.3 N; T1: 62.1 N) and superior springiness and cohesiveness in T3, confirming that the instrumental and hedonic texture assessments are mutually consistent. The superior springiness of T3 paneer appears to translate directly into a perceptibly more elastic, bouncy mouthfeel that panellists found comparable to or better than the control [21,59].

T2 (0.5%) achieved acceptable but undistinguished scores across all attributes, consistently intermediate between T1 and T3. At this inclusion level, the concentration of flavour-active volatile compounds is insufficient to generate the synergistic aroma enhancement observed in T3, while the green colour shift (a* = −5.16) is more pronounced and was perceived as less attractive than T3’s warmer green-yellow. This combination of insufficient flavour benefit and excessive colour deviation explains T2’s lower overall acceptability (6.6) relative to both T1 (7.7) and T3 (8.2).

T4 (1.5%) showed significantly depressed scores for aroma (5.8), body and texture (5.8), flavour (6.1), and taste (6.0) all falling substantially below the control. These parallel declines are mechanistically explained by two intersecting factors. First, the elevated concentrations of quercetin, kaempferol, and condensed tannins at 1.5% inclusion exceed the sensory detection thresholds for bitterness (approximately 50 µg/g for quercetin [59]) and astringency which were confirmed at 67.8 µg/g quercetin by HPLC in T4, producing perceptible off-flavour notes through formation of stable complexes with salivary proline-rich proteins and mucin glycoproteins, reducing salivary flow and producing a drying, puckering mouthfeel [33,60,61]. Second, the substantially reduced hardness (48.7 N) and cohesiveness (0.61) in T4, both significantly lower than T1, translate into an excessively soft, crumbly texture that panellists found unsatisfying. Despite this, overall acceptability in T4 (7.0) remained above the midpoint of the hedonic scale, indicating the product was not unacceptable but it was significantly inferior to T3 [29,32].

Taken together, the sensory results of this study confirm that T3 (1.0% S. grandiflora leaf powder) offers the best overall sensory–technological balance among the treatments tested, combining strong acceptability with a favourable—though not the highest—nutritional profile (T4 showed higher protein, fibre, phenolic, flavonoid and antioxidant values), the most favourable texture profile among the treatments tested, with microbial counts generally comparable to or better than the other fortified treatments. The convergence of sensory, instrumental, microbiological, and nutritional evidence in favour of T3 provides a multi-dimensional, scientifically robust foundation for the recommendation of 1.0% S. grandiflora leaf powder as the target commercial fortification level for this novel functional paneer product.

4. Conclusions

Incorporation of 1.0% Sesbania grandiflora leaf powder (T3) into paneer significantly improved its nutritional and functional properties compared with the unfortified control. Fortification increased protein (21.00%), crude fibre (2.10%), total phenolic content (4.07 mg GAE/g dw), total flavonoid content (1.25 mg RE/g dw), and antioxidant activity (DPPH IC₅₀ = 6.15 mg/mL). HPLC analysis identified quercetin and kaempferol as the predominant bioactive compounds contributing to the enhanced antioxidant potential. The reduction in water activity (0.958) and the presence of phenolic compounds contributed to improved microbial quality, with total plate counts remaining within BIS acceptable limits throughout 9 days of refrigerated storage. However, yeast and mould counts exceeded the recommended BIS limits in all treatments, including T3, by day 9, indicating that further improvements are required to extend microbiological shelf life. Texture profile analysis and SDS-PAGE results suggested the incorporation of plant proteins into the paneer matrix, although structural integration within the casein network was not confirmed. Sensory evaluation identified T3 as the most acceptable formulation, achieving the highest overall acceptability (8.2/9) and flavour score (8.5/9). Overall, 1.0% Sesbania grandiflora leaf powder represents the optimum fortification level for producing nutritionally enriched, antioxidant-rich, and sensorially acceptable functional paneer. Future research should validate these findings through expanded sensory evaluation, direct mineral quantification, bioaccessibility and bioavailability studies, shelf-life validation under commercial processing conditions, and assessment of large-scale production feasibility before commercial application [17].