Microchemical Journal 204 (2024) 111068

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Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

pH-indicator based on delignified jute fiber and red cabbage anthocyanins

for monitoring fish spoilage using a smartphone application
Chaithra K P , Sonia Theresa Benjamin , Vinod T.P. *
Department of Chemistry, CHRIST (Deemed to be University), Hosur Road, Bengaluru 560029, India

A R T I C L E I N F O A B S T R A C T

Keywords: Halochromic materials that show visible color changes in response to changes in pH are suitable for the real-time
Red cabbage anthocyanins monitoring of fish spoilage. In this study, an easy-to-use, simple, inexpensive, and non-toxic fish freshness in­
Jute fiber dicator was fabricated by combining delignified jute (Corchorus olitorius) fibers and anthocyanins (halochromic
Delignification
materials) from red cabbage (DFA: Delignified jute fibers incorporated with anthocyanins). A single-step
Fish freshness monitoring
Smartphone-based detection
decolorization/delignification using solar irradiation along with NaOH and H2O2 treatment was used for
modifying the jute fibers. This method helps to overcome the self-color, mitigates the lack of affinity of jute fibers
towards anthocyanins and preserves the lignin so that the strength of the fiber is not impacted. A smartphone-
based color analysis was used for real-time fish quality monitoring using DFA. To the best of our knowledge,
there are no reports on the use of jute fibers as substrates to incorporate anthocyanins for food spoilage moni­
toring. The indicator displayed an observable color response to the pH and varying concentrations of amine
compounds. During the storage of fish (mackerel), the colorimetric indicator showed a visible color change from
pink (for fresh fish) to blue (for spoiling fish) and then to green (for spoiled fish), corresponding to changes in pH
and total volatile basic nitrogen. To offer a straightforward quantitative assessment of color changes, we utilized
the freely available Android application Color Grab to measure the color using RGB and L*, a*, and b* indices.
The DFA indicator providing naked-eye analysis has the potential to be an effective tool for real-time monitoring
of on-site food spoilage by non-specialized personnel in resource-limited areas.

1. Introduction risks.
Spoilage in fish occurs as a result of microbial activity in fish muscle
Food waste is a major global challenge not only from an ethical and and leads to biochemical changes, including the formation of nitroge­
social point of view, but also from environmental and economic ones nous gases due to the degradation of proteins and lipids [8]. The
[1]. An estimated 17 % of total global food production is wasted (in breakdown of proteins results in the generation of total volatile basic
which 11 % is in households) [2]. Among the various globally consumed nitrogen (TVB-N) compounds, such as, dimethylamine (DMA, formed by
perishable foods, fish is an important category related to nutrition, autolytic enzymes during frozen storage), ammonia (NH3, generated
contributing approximately 17 % of animal protein consumption [3]. through nucleotide catabolites and deamination of amino acids), and
Additionally, fish play a crucial role in supplying essential micro­ trimethylamine (TMA, resulting from the breakdown of a crucial fish
nutrients, vitamins, and essential fatty acids, addressing nutritional molecule, trimethylamine N-oxide, involved in osmoregulation, consti­
deficiencies in the diets of economically underprivileged populations tuting a significant portion of TVB-N during spoilage), resulting in an
[4]. Fish wastage is more prominent in the consumer level during the increase in pH [9–11]. Changes in fish quality can be monitored by
edible (fresh) and inedible (spoiling or spoiled) consumption stages. In measuring the pH during storage and this can serve as an indicator of
recent years, there has been growing interest in the development of fish quality [10,12,13].
simple and cost-effective techniques for real-time monitoring of fish Colorimetric indicators derived from natural sources are suitable for
freshness [5–7] to ensure food safety, food quality, and reduction of food monitoring fish quality [11,14–18]. They offer benefits such as cost-
waste. Rapid and accurate assessment methods are essential in pre­ effectiveness and easy detection of color changes, both quantitatively
venting the consumption of spoiled fish which can pose serious health (via imaging software) and qualitatively (direct observation with the

* Corresponding author.
E-mail address: vinod.tp@christuniversity.in (V. T.P.).

https://doi.org/10.1016/j.microc.2024.111068
Received 7 April 2024; Received in revised form 1 June 2024; Accepted 24 June 2024
Available online 24 June 2024
0026-265X/© 2024 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.
C. K P et al. Microchemical Journal 204 (2024) 111068

naked eye) [7]. Natural materials that show visible color changes in 2. Materials and methods
response to changes in pH are halochromic indicators that comprise
halochromic dyes and support [11,19]. Natural halochromic dyes, such 2.1. Materials
as anthocyanins, alizarin, curcumin, and shikonin, are easily available,
accessible, non-toxic and biodegradable [20–24]. Among these natural The commercially available jute (Corchorus olitorius) fiber yarn was
pH-sensitive dyes, anthocyanins have been widely used for monitoring obtained from a local village in Kerala, India. Fresh Indian Mackerel
fish quality. In particular, anthocyanins extracted from red cabbage are (Rastrelliger kanagurta) was collected from a fish supermarket (deceased
favoured for fish quality assessments [11,18,25–28]. This preference is and stored in ice) on the same day as the supplier’s delivery and red
due to their ability to exhibit a rose-red to pinkish-purple color under cabbage was collected from a local supermarket in Bangalore, Karna­
acidic conditions, purple-blue color at neutral pH, green to yellowish- taka, India. Hydrochloric acid (HCl), ethanol, NH3, TMA, and DMA from
green in alkaline environments [11,29,30] and resistance to heat and Sigma Aldrich. Sodium dihydrogen phosphate (NaH2PO4), disodium
light compared to anthocyanins extracted from other natural sources hydrogen orthophosphate (Na2HPO4), boric acid, potassium carbonate
[31,32]. (K2CO3), bromocresol green, methyl red, trichloroacetic acid (TCA)
The literature on anthocyanin-based fish freshness indicators is were obtained from SD Fine-Chem Pvt Ltd., India.
compiled and compared in Table 3. Publications point that various
substrates used to incorporate anthocyanins include poly (ε-capro­ 2.1.1. Choice of materials and background
lactone) [32], polyvinyl alcohol [18], chitosan [33], sodium alginate The jute fiber and red cabbage are inexpensive, easily available (even
[25] etc. The preparation of these polymer solutions and the methods in resource-limited areas), biodegradable, and eco-friendly. The jute
used to fabricate the indicator are time consuming, and the substrates fiber is a natural fiber abundantly available without any impact on food
are not readily available in resource-limited settings. To address these production. It has been used, since the 19th century [1,2] for various
limitations, in the current work, we employed a readily available, commercial and industrial purposes, including food packaging [3]. The
relatively low cost, biodegradable, and low environmental impact ma­ fish chosen for analysis was Indian Mackerel (Rastrelliger kanagurta),
terial – natural jute (Corchorus olitorius) fiber. Single-step decoloriza­ which is one of the widely consumed marine fishes in the South-East
tion/delignification was used to modify the jute fibers, helping in Asian countries. It contains various nutrients which are of high signifi­
overcoming the self-color and lack of affinity towards anthocyanins cance to the human health [45,46]. Indian Mackerel is rich in poly-
[34]. It was used as a substrate for incorporating anthocyanins. The unsaturated fatty acids which are commonly known as long chain
present work also employed an accessible, equipment-free, and user- omega-3 fatty acids [45,47]. There have been no studies in the literature
friendly smartphone-based free application to quantify the color on Indian Mackerel food quality monitoring using fibers incorporated
changes in response to pH changes to alleviate the use of colorimeters with halochromic indicators.
[32,35], and computer-aided software (Photoshop [35], CIELAB [12],
ImageJ [33], etc.), which are used to quantify the color change shown by 2.2. Preparation of the indicator
the indicator, which requires expertise and training.
Jute fibers (JF) are one of the cheapest, renewable, and most 2.2.1. Delignification of the JF
commercially accessible fiber with a low-carbon food print and high JF mainly consists of three principle constitutes α-cellulose, hemi­
toughness among all natural fibers [36,37]. Jute is extensively culti­ cellulose, and lignin [40], of which- chromophore groups in lignin are
vated in Southeast Asian countries such as India, Bangladesh, Myanmar, responsible for the brown color [42], making the color changes in the
China and Nepal [38], and it has applications in packaging, textiles, proposed indicator indistinguishable in response to pH (Fig. S1, Sup­
furniture, construction, and automobile [39]. JF mainly consists of three porting information). Different methods for the delignification include
principle constitutes α-Cellulose, hemicellulose, and lignin [40,41], of the use of alkaline Na2SO3, NaClO2, and NaClO [48]. These methods are
which chromophore groups in lignin are responsible for the brown color time-consuming and require a large volume of toxic chemicals, and the
[42]. The delignification of jute fibers enhances their porosity, removes by-products are difficult to dispose of [42]. The delignifying agents
the lignin, and makes the fiber white. Delignified jute fiber (DF) is an NaOH and H2O2 were chosen over other chemical treatments in this
ideal substrate with the ability to adsorb dyes for incorporating antho­ study because they are easily available, less toxic, and environmentally
cyanins through a dip-coating method for monitoring fish quality. benign, as they produce oxygen and water as by-products [49,42,50].
Compared to various reported methods (solution casting [35], 3D The process of delignification involves the application of 2.5 M
printing [6], electrospinning [32], spin coating [43], sol–gel [44], etc.) NaOH onto 15 cm of JF, followed by dipping in 30 % H2O2. JF was then
(Table 3) to incorporate anthocyanins, directly dipping or soaking the exposed to sunlight (Fig. 1) until a white jute fiber was obtained (Fig. S2,
indicator solution into the substrate followed by solvent evaporation is Supporting information). The delignification process of JF was opti­
simple, inexpensive, and requires relatively less time [12]. To the best of mized by varying (i) the amount of NaOH (1–4 ml), (ii) amounts of H2O2
our knowledge, there is no existing literature on the use of jute fibers as (1–4 ml), and (iii) time intervals of exposure to sunlight (15 min to 60
substrates to incorporate anthocyanins for food spoilage monitoring min) to produce white jute fiber (DF) with good tensile strength. The
through smartphone-based analysis. obtained fiber was subjected to multiple washes with distilled water to
In the present study, a pH-sensitive delignified jute fibers incorpo­ remove any additional residues and solvents. The jute fiber obtained
rated with anthocyanins (DFA) was designed by leveraging the com­ after delignification is referred to as the DF. Delignification can be
bined properties of DF and red cabbage anthocyanins, followed by conducted under visible light from a tungsten lamp (75 W) in countries
smartphone-based color analysis for monitoring fish quality. Color where sunlight exposure is very low, and the use of this light reduces the
changes were quantified using the L*, a*, b*, and RGB values. The in­ exposure time by half (15 min). The color response of the DF to pH 2.00
dicator was used to monitor the quality of mackerel fish, and the color was monitored after incorporation with anthocyanins after each opti­
change was quantified by simultaneously measuring TVB-N and pH mization (Fig. S3, Supporting information). The optimized treatment
changes. The integration of halochromic materials and smartphone conditions to obtain DF that showed a naked-eye distinguishable color
application enhances the user-friendly nature of the system, making it change in response to pH 2.00 were 1 ml NaOH, 2 ml H2O2, and 30 min
practical for widespread use in various settings. of sunlight exposure for 15 cm of JF.
Under alkaline conditions, H2O2 dissociates to form the hydro­
peroxyl anion (OOH–), which oxidizes the chromophores in lignin
through the cleavage of side chains, resulting in decolorisation without
complete removal of the lignin structure [42,51] (H2O2 + OH– ↔ OOH–

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C. K P et al. Microchemical Journal 204 (2024) 111068

Fig. 1. Schematic illustration of the process of delignification of jute fiber and preparation of halochromic indicator.

+ H2O, OOH– + chromophore → bleaching (chromophore destroyed)

I002 − Imin
[52]). The NaOH pre-treatment selectively separate lignin from carbo­ CI = × 100 (1)
I002
hydrates, thereby increasing the relative amounts of cellulose and
hemicellulose [53,51] resulting in the white jute fibers with enhanced Where, I002 is the maximum peak intensity (2θ) between 22◦ and 23◦
porosity, dye affinity, and absorbability [54]. This method effectively and Imin is the minimum peak intensity between 18◦ and 19◦ for cellu­
fixes the anthocyanins to the jute fibers and preserves the lignin so that lose I.
the strength of the fiber is not impacted [34].

2.4. Physical properties of the fibers

2.2.2. Freshness indicator (DFA) fabrication
Anthocyanins were extracted from the red cabbage (Brassica oleracea
The tensile strength (TS) were evaluated by fixing the fiber to a
L.) using a previously reported method [55]. In brief, ~150 g of red
tensile strength analyzer (MX-500 N, IMADA,) at a crosshead speed of
cabbage was crushed, macerated, and then added to an ethanol–water
10 mm/min. Moisture Content (MC), and Water Solubility (WS) of the
mixture (80 ml) at a volume ratio of 7:3. HCl (1.00 mM) was added to
DF and DFA were measured in triplicate based on the reported methods
adjust the pH to 2.00. The mixture was stored at 4 ◦ C for 24 h, after
[26,58]. A complete description of this method is provided in the Sup­
which the supernatant was centrifuged at 2000 rpm for 10 min and
porting Information.
vacuum-filtered. The obtained anthocyanin extract was stored in a dark
The thermal stability of the fibers was examined using thermogra­
bottle at 4 ◦ C until its use. The total anthocyanin content (TAC) was
vimetric analysis (TGA; PerkinElmer STA 6000). The analysis was per­
calculated using the pH differential method, an AOAC method (2005.02)
formed at a heating rate of 10 ◦ C/min under a nitrogen flow rate of 20
[56,57]. A description of this method is given in the Supporting
mL/min. Samples of 6–12 mg each were analyzed from 30 ◦ C to 800 ◦ C.
Information.
The DFA indicator was prepared by dipping 15 cm of DF into ~10 ml
of anthocyanin solution for 15 min. The fiber was then dried for 15 min 2.5. pH, DMA, TMA, and NH3 sensitivity of DFA
and cut into small pieces (3 cm), as shown in Fig. S4 (Supporting
Information). To investigate the halochromic behavior of the indicator, the color­
imetric response of the DFA indicator to various phosphate buffers
(NaH2PO4 and Na2HPO4) of pH ranging from 2.00 to 12.00 were pre­
2.3. Characterization of the fibers pared. The pH of the buffer solution was monitored using a Hanna pH
meter (Pocket pH Tester with 0.01 pH Resolution – HI98108). At each
An optical profilometer (Zeta-20 Optical Profilometer, Zeta In­ pH point, 20 µL of solution was added to the DFA indicator. After in­
struments) was used to examine the morphological features and cubation for 2 min at room temperature, the color change was photo­
roughness of the JF, DF, and DFA. Attenuated total reflection with graphed using a smartphone camera.
Fourier-transform infrared spectroscopy (ATR-FTIR, Shimadzu IR Spirit- The effectiveness of a fish freshness indicator depends on the mini­
L) was used to study the functional groups available in JF, anthocyanins, mum quantity of amines and the minimum duration necessary to show a
and the modified groups after delignification and incorporation of an­ color change. To examine the color response sensitivity of the DFA in­
thocyanins. Modifications in the morphological and physical properties dicator to various concentrations of NH3, DMA, and TMA, the TVB-N
of JF and DF were analyzed using scanning electron microscopy (SEM, sensitivity was tested according to a previously reported method [59].
Apreo S Lovac). The crystallinity of JF and DF was determined using X- Ammonia solutions were prepared at various concentrations ranging
ray diffraction (XRD, Rigaku Miniflex X-ray diffractometer). The crys­ from 0.0025 M to 1.00 M. 125 ml of each solution was poured into a
tallinity percentage was calculated using the following Eq. (1) [38], closed container, and the indicator attached onto the cap of the

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C. K P et al. Microchemical Journal 204 (2024) 111068

container (1 cm above the solution) was exposed for 30 min at 4 ◦ C, and (IBM SPSS Statistics 29.0, Chicago, IL, USA). The mean values were
one sample was exposed to distilled water as a control sample. The color compared using one-way analysis of variance and Duncan’s multiple-
changes in the DFA indicator were captured immediately after 30 min of range test at p < 0.05 significant level.
exposure to ammonia. The same procedure was used to examine the
sensitivity of the DFA indicator towards DMA and TMA. 3. Results and discussion
To evaluate the sensitivity of the indicator to ammonia over time, a
sealed vessel was filled with 150 ml of a 0.005 M ammonia solution. The 3.1. Characterization of the fibers
DFA indicator was positioned 1 cm above the solution inside a container
at 25 ◦ C. Photographs of the indicator color changes were taken at 10 to The morphology, surface roughness, and textural features of the JF
60-second intervals. and DF were analyzed using an optical profilometer. The 2D microscopic
images of JF and DF are shown in Fig. S8a, and S8b and the 3D images in
2.6. Color analysis Fig. S8d, and e (Supporting Information), respectively. The 2D images of
JF and DF showed no changes in the morphology and textural features
The color response of the indicator to TVB-N, pH, and spoilage were after delignification. The white color of the DF in the 2D images con­
photographed using a smartphone camera (Vivo V11 Pro) at a distance firms the delignification of JF. The delignification was further confirmed
of 5 cm against a white background with vertical lighting and a fixed by the reduced values of the surface roughness from Sa (average abso­
angle. The captured images were analyzed using a cost-free Android lute deviation of the surface), 233.55 ± 3.65 µm (JF) to 64.32 ± 2.03 µm
app, Color Grab [60] from Loomat Developers, to measure the R (Red), G (DF) [Table S1, Supporting Information] measured at three different
(Green) and B (Blue) and L* (Lower values refer to darkness, and higher areas of the same sample.
values indicate lightness), a* (where positive values refer to red and ATR-FTIR analysis was performed on the JF and DF to identify the
negative to green), b* (where positive values indicate yellow and functional groups present in JF, and the modified groups formed after
negative blue) values as shown in Fig. S5 (Supporting Information). A delignification (Fig. 2a). JF showed a band at 3200–3600 cm− 1, corre­
step-by-step analysis is provided in the Supporting Information. sponding to the O–H stretching vibrations of cellulose and hemicellu­
The color of the DFA indicator was quantified with L, a, and b using lose. The band at 2900 cm− 1 was due to the C-H symmetrical stretching
the total color difference (ΔE) [61] and RGB indices using the response of hydrocarbons in cellulose, hemicelluloses, and lignin units. The band
sensitivity (S) [62]. The equations used for the quantification are as at 1509 cm− 1 was due to C=C aromatic stretching vibrations, 1242 cm− 1
follows: was due to C-O stretching, and 1050 cm− 1 was due to aromatic C–H in-
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ plane deformation of lignin [38,65,66]. The signals corresponding to
ΔE = (L* − L)2 + (a* − a)2 + (b* − b)2 (2) lignin (1509 cm− 1 and 1050 cm− 1) weakened in DF but did not
completely disappear, indicating the removal of chromophore groups in
L* a* and b* were the color parameters of the indicator, which were lignin without the complete removal of the lignin structure.
measured before the analysis and were taken as standard and L, a, and b The XRD patterns were recorded to understand the effect of
were the color parameters of the indicator after its color change in delignification on the crystallinity of the DF. The diffractogram (Fig. 2b)
response to various pH values or volatile amines. revealed peaks at 2θ = 15◦ , 22.5◦ , and 34.5◦ , assigned to the (1 1 0),
(2 0 0), and (0 0 4) planes, respectively, corresponding to the amorphous
|R* − R| + |G* − G| + |B* − B| and crystalline regions of the cellulose I structure [67,38] in the JF and
S(%) = × 100 (3)
R+G+B DF. The increase in the peak intensity corresponding to the (2 0 0) plane
and peak shift in DF indicates an increase in crystallinity and the
R* G* and B* were the color parameters of the indicator and RGB were
transformation of cellulose Iβ into cellulose Iα due to the alkali treatment
the color parameters of color response in indicators.
[67,68], respectively. The percentage of crystallinity increased from
57.76 % of JF to 65.42 % of DF due increase in the crystallinity of the JF
2.7. Application of DFA indicator in real-time freshness monitoring of fish after delignification. The SEM images of JF and DF are shown in Fig. 2c-
f. The morphology and size (~30 µm, Fig. S9, Supporting Information)
2.7.1. Fish spoilage trial of the fibers remained the same even after delignification. The surfaces
The fish spoilage analysis was conducted using the Indian Mackerel of the DF were smoother than those of the raw fibers. The tensile
(Rastrelliger kanagurta). The fish pieces of the similar weight (50 g) and strength of the JF and DF was 450.01 ± 5.50 N/mm2 and 387 ± 3.87 N/
similar sizes were placed in a closed plastic containers at 25 ◦ C and 4 ◦ C mm2 (Fig. S10, Supporting Information). The decrease in the tensile
to monitor their freshness. The DFA indicator was stuck at the top of a strength of the DF is due to the removal of lignin, which acts as a binder
transparent closed container lid against a white background to distin­ for micro-fibrils in JF [54]. However, DF has a significant tensile
guish visible color changes (Fig. S6a, Supporting Information), and was strength and can act as a substrate to incorporate anthocyanins.
also positioned 3 cm away from the sample and 2 cm from the bottom of
the container (Fig. S6b). The images of the DFA were captured and 3.2. DFA indicator and its physical properties
quantified at various intervals time at room temperature and 4 ◦ C.
The pH and TVB-N changes during the storage of fish were deter­ The morphology and textural features of the DFA indicator remained
mined at regular intervals, along with their ΔE value at the same time. the same after incorporation of anthocyanins, except for a color change
For pH measurement, 5 g of fish (Indian Mackerel) flesh was homoge­ from white to rose-red (Fig. S8c, Supporting Information). The FTIR
nised in 50 ml of distilled water and pH was quickly monitored using the spectra of DF, anthocyanins, and DFA are shown in Fig. S11, Supporting
Hanna pH meter. The TVB-N analysis was carried out using Conway information. The spectrum of anthocyanins showed a broad band at
diffusion method (Fig. S7, Supporting information) [63] according to 3200–3400 cm− 1 and at 1043 cm− 1 corresponding to the –OH group and
FAO, 1979 [64]. A description of TVB-N analysis is provided in the aromatic ring C-H deformation, respectively. The band at 2983 cm− 1
Supporting Information. was assigned to the C–H stretching vibration of the aromatic rings. The
bands at 1626 and 1430 cm− 1 are due to the stretching vibrations of the
2.8. Statistical analysis C=C aromatic ring. The spectrum of the DFA indicator showed changes
in intensity and overlapping bands of DF and anthocyanins, indicating
The results, presented as mean ± standard deviation, were assessed the incorporation of anthocyanins into the DF substrate. The TAC was
in triplicate and subjected to statistical analysis using SPSS software found to be 116.82 ± 2.86 mg/L.

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Fig. 2. a) FTIR, b) XRD Spectra and SEM image of JF (c, d), and DF (e, f).

The physical properties of the films, such as moisture content (MC) H2O2/NaOH treatment of the jute fibers preserved lignin and its strength
and water solubility (WS), are presented in Table S2. The moisture [34]. Moreover, the onset temperature (Tonset) for hemicellulose and
content values were very low, and there was no significant change (p < offset temperature (Toffset) for cellulose slightly shifted slightly for DF
0.05) in either the DF or DFA. However, the water solubility slightly and DFA compared to the JF, data in Table S3. The results suggest that
increased in DFA (5.76 %) compared to DF (3.33 %), which was due to DFA has excellent thermal stability below 100 ◦ C and this indicator is a
the water-soluble nature of the anthocyanins extracted from red cab­ promising option for food monitoring.
bage. The increase in solubility of the DFA indicator was not compara­
tively higher, indicating its water resistance ability and structural 3.3. Color response of the DFA at different pH
integrity when exposed to pH changes during food spoilage. This pre­
vents dye migration to the food sample, making DFA an ideal pH Color and visual characteristics decide the suitability of halochromic
indicator. indicators for consumer use. The substrate, DF, was colorless with color
The thermal stabilities of JF, DF, and DFA were investigated using parameters of 232 ± 3.21 (R), 230 ± 4.31 (G), and 205 ± 5.61 (B), and
TGA, which revealed the changes in biomass weight due to physical and the L* value 90.95 ± 1.35, b* value of 12.49 ± 0.41, and a* value of
chemical variations at a specific heating rate. The weight of the samples − 2.42 ± 0.21. The DFA was rose-pink in appearance with an L* value of
decreased with increasing temperature owing to the devolatilization of 54.2 ± 0.87, b* value of − 3.2 ± 0.21, and an increased a* value of 55.5
moisture and organic content. Fig. S12 (Supporting information) shows ± 0.45 in comparison to the DF. This observation validates the presence
the TGA and derivative thermogravimetric (DTG) curves of JF, DF, and of anthocyanins in the DF. The L*, a*, b*, and RGB values of the DFA
DFA. The curves indicate distinct stages of thermal degradation corre­ indicator served as reference parameters for all the analyses.
sponding to the presence of lignin, hemicellulose, and cellulose, each The color response of the DFA indicator to pH changes from 2.00 to
degrading at different temperatures [38]. The first stage was from 30 to 12.00 was evaluated using phosphate buffer. Table 1 indicates that DFA
145 ◦ C, with 15–20 % weight loss corresponding to the evaporation of changes its color from rose-red to pinkish-purple as the pH level changes
volatile chemicals and moisture from all the samples. The second stage from acidic (pH 3.00–6.00) to neutral (pH 7.00) and then to purple-blue
occurred at 150–250 ◦ C, possibly because of the degradation of hemi­ from pH 8.00–10.00. The purple-blue color turns green or yellowish-
cellulose [34]. The third stage occurred from 250 to 550 ◦ C, with the green at basic pH (pH 10.00–12.00). The color response obtained was
most substantial weight loss occurring from 270 to 350 ◦ C due to cel­ consistent with that previously reported in the literature [24,70]. These
lulose and from 270 to 550 ◦ C due to lignin decomposition [34,38]. The color changes were in agreement with the L*, a*, and b* color param­
last stage at higher temperatures was due to lignin decomposition eters. At acidic pH, higher and more positive a* values indicate red,
continuing until 800 ◦ C and the aromatization of a few cellulose residues while at pH 7.00 to 9.00, positive a* and negative b* values indicate
[69]. The Tpeak (◦ C) for all the fibers remained the same, indicating that purple and purple-blue color, respectively. The negative b* and a*

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Table 1 values at basic pH indicate blue-green and green color. The color
Colorimetric parameters of the DFA indicator and its representative images in changes observed in the DFA in response to changes in pH were due to
response to different pH solutions.* structural changes in cyanidin-3-glucoside (anthocyanins) in red cab­
pH Images of DFA L* a* b* ΔE bage (Fig. S13, Supporting Information). This compound transforms
value indicator from a red flavylium cation at a pH of 2.0–3.0 into a pinkish-purple
2 54.23 ± 49.57 ± 4.43 ± 9.77 ± quinonoidal base at a pH of 4.00–6.00, owing to rapid proton loss. At
1.06c 1.66b 0.75a 2.06g pH 7.00, the structure shifted to purple quinoidal anhydrobase, followed
by blue-ionized quinoidal anhydrobase at pH 8. An increase in pH to
>10 leads to the formation of green and yellow chalcones [24,71,72].
The indicator showed visible color changes in response to pH with most
3 50.83 ± 60.10 ± 3.27 ± 8.84 ± of the ΔE values falling within the range of 6.00–12.00 indicating a color
0.57c 1.71a 0.64a 1.16gh change that is noticeable to the human eye [73]. In addition, the ΔE of
the DFA indicator had a good linear relationship with pH 4–10 (y =
6.91x + 22.56, R2 = 0.975), and the limit of detection (LOD) was
determined to be 0.71, suggesting the appropriate sensitivity (LOD =
4 51.50 ± 55.76 ± 1.70 ± 5.65 ± 3.3 K/N, where K is the standard deviation of blank measurements and N
0.35c 0.75a 1.08a 1.16h is slope of the calibration curve).
As shown in Fig. S14a (Supporting Information), the absorption peak
was observed at ~520 nm for pH <5.00, corresponding to the red fla­
vylium cation [74]. At pH 6.00 and 7.00, the maximum absorption peak
5 59.11 ± 42.06 ± − 6.11 ± 14.42 ± gradually shifted from 520 to 550 nm, accompanied by a decrease in
0.61b 1.87c 1.06b 1.07f absorption, which can be attributed to the carbinol pseudo-base
(colorless) and purple quinoidal anhydrobase [75]. The absorption in­
tensity showed a bathochromic shift from 550 to 600 nm for pH 8.00 to
10.00, possibly due to the quinoidal base (blue). The change in the
6 43.87 ± 40.87 ± − 6.27 ± 18.05 ± maximum absorption peak at pH 11 and 12 can be attributed to the
0.51d 1.29c 0.94b 1.59e accumulation of green and yellow chalcones, respectively [76]
(Fig. S14b, Supporting Information).

3.4. NH3, DMA, and TMA sensitivity of DFA

7 53.03 ± 36.33 ± − 18.07 ± 24.15 ±
0.40c 1.99d 1.76e 1.06d TVB-N has been used as an important marker for determining the
extent of fish spoilage. Anthocyanins present in DFA can exhibit color
changes owing to the basic environment generated by the diffusion of
TVB-N compounds. The minimum amount of amines and the time
8 58.00 ± 58.17 ± − 26.93 ± 29.65 ± required to produce a color change in the indicator determines the
1.17b 0.66a 1.64g 1.09d ability of the indicator to act as a fish spoilage indicator.
As shown in Fig. 3a, the indicator changed from pink to blue and
finally green when exposed to ammonia solution (0.005 M) within 60 s.
The initial color change was observed within 10 s. The response sensi­
9 65.40 ± 25.00 ± − 23.67 ± 38.31 ± tivity S (%) and the total color difference (ΔE) values for the color
1.59a 1.55e 1.25g 1.86c changes increased with exposure time (Fig. 3b). The DFA indicators also
exhibited visible color changes from pink to blue to green to yellow
(Fig. 3c) when exposed to different ammonia compounds (NH3, DMA,
and TMA), at concentrations ranging from 0.0025 M to 1.00 M. The ΔE
10 67.97 ± − 7.47 ± − 22.33 ± 53.33 ± values increased from 30 to 80 with an increase in ammonia concen­
0.42a 1.66f 0.60f 0.77b
tration, with decreased values at high concentrations owing to the
yellowish-green color. In addition, the ΔE of the DFA indicator had a
notable linear relationship with ammonia concentration (10–100 μmol/
L, y = 0.27x + 54.96, R2 = 0.987), and the limit of detection (LOD) was
11 43.30 ± − 18.97 ± − 14.10 ± 75.90 ± determined to be 1.63 μmol/L, indicating high sensitivity of the pre­
1.71d 1.27g 1.06d 0.71a
pared indicators to volatile nitrogen compounds. The naked-eye
detectable color change at the minimum concentration of 0.0025 M,
equivalent to 17 mg NH3/100 ml (TVB-N evolved during the spoiling
stage of the fish [59]), and visible color change within a few seconds
12 35.63 ± − 19.53 ± − 5.07 ± 74.55 ± indicated that the DFA indicator could be considered as a useful indi­
1.89e 0.66g 1.21c 2.06a
cator of fish freshness.

3.5. Real-time fish freshness monitoring

*
Different superscript alphabets in each column indicate significant differ­
The developed DFA indicator was used to monitor the spoilage of
ences (p < 0.05).
mackerel fish by detecting color changes during storage at room tem­
perature and in a refrigerator (4 ◦ C). Anthocyanins present in the indi­
cator show color changes in response to volatile amines produced by the
biochemical and microbial activity of the fish upon spoilage. Changes in
mackerel quality were confirmed using TVB-N values. According to

6
C. K P et al. Microchemical Journal 204 (2024) 111068

Fig. 3. a) Photographs and b) quantification of color response in the DFA indicator upon exposure to ammonia solutions with time, and c) the color change of the dfa
indicator when exposed to nh3, DMA, and TMA with concentrations ranging from 0.0025 M to 1.00 M.

Table 2
Photographs and color parameters of the DFA indicator upon fish spoilage with TVB-N, and pH values on different days of storage at 4 ◦ C.
Time Color of DFA Freshness ΔE S (%) TVB-N pH
(days) (mg N/100 g)

0 20.57 ± 2.11d 32.21 ± 3.61f 5.6 ± 0.57f 6.80 ± 0.02d

2 38.83 ± 1.93c 85.18 ± 3.29d 8.4 ± 0.71e 7.14 ± 0.03c

4 67.33 ± 1.41b 79.09 ± 3.41e 14.13 ± 1.10d 7.56 ± 0.01b

6 69.09 ± 0.57b 131.36 ± 4.47c 20.61 ± 2.01c 8.14 ± 0.02a

8 77.04 ± 0.60a 274.66 ± 5.36b 28.19 ± 2.11b 8.56 ± 0.03a

10 78.43 ± 1.10a 388.01 ± 0.59a 35.33 ± 2.83a 8.78 ± 0.04a

7
C. K P et al. Microchemical Journal 204 (2024) 111068

previous reports, mackerel fish were considered spoiled if their TVB-N quantitatively.
values were 25 mg N/100 g or more [32,77]. These changes, along In comparison to other reported fish freshness indicators prepared by
with the pH and indicator chromaticity values, are listed in Table 2. incorporating pH-sensitive anthocyanins from red cabbage (Table 3),
The TVB-N and pH values suggest that the mackerel fish sample the current study (i) reduced the preparation time of indicator to 40 min,
(Fig. S13, Supporting Information) was fresh during the initial two days (ii) used commercially available biodegradable jute fiber as a substrate,
of storage at 4 ◦ C, with values less than 10 mg N/100 g and very low pH and (iii) used non-computer-aided free Android software for color
changes. The steady increase in TVB-N (<25 mg N/100 g) and pH values analysis. Substrate preparation only requires jute fibers with delignifi­
during 4–6 days of storage indicates the fish use now/spoiling state. The cation, which can be performed within 30 min in sunlight without any
pH level increased during this period because of the formation of TMA equipment. Delignification enhances porosity, dye affinity, and
due to the degradation of TMAO. After seven days of storage, the fish absorbability. The dip-coating method used for the indicator prepara­
samples were considered spoiled as the TVB-N level exceeded the tion is simple and requires less time. The white color of the DF makes the
rejection limit of 25 mg N/100 g. The increased pH values during the color change more distinguishable.
spoiled stage were due to the formation of NH3 and other volatile
amines, even after the depletion of TMAO [9].
3.6. Reversibility and stability of the indicator
The DFA indicator used to monitor spoilage of mackerel fish showed
noticeable and measurable color changes in correlation with TVB-N
The reversibility of the DFA indicator was assessed by adding solu­
values. The color changes ranged from pink (for fresh fish) to blue (for
tion of pH 2.00 to a pH 12.00 solution at 25 ◦ C. This procedure was
spoiling fish) and then to green (for spoiled fish). The RGB, S (%), L*, a*,
repeated ten times. As shown in Video S1 (Supporting Information), DFA
b*, and ΔE values of the DFA indicator for the observed color changes
showed the same pink and green color after ten cycles of repeated
are recorded in Table S3 (Supporting Information). The L* and a* values
addition of pH 2.00 and pH 12.00, respectively, and maintained their
were higher and more positive during the first two days of storage,
original pH-sensing capability after repeated exposure to acidic and
indicating a bright pink color. The b* (4–6 days) and a* (8–10 days)
basic pH, indicating that the DFA fibers could be reused.
values were highly negative during later days of storage, indicating blue
The stability of the DFA indicators was monitored by storing them at
and green color, respectively. The ΔE values increased from 20.57 ±
4 ◦ C and 25 ◦ C in a sealed container after fabrication. Photographs of the
2.11 for fresh fish to 67.33 ± 1.41 for spoiling fish and eventually to
color change in response to pH 7.00 were captured at various time in­
78.43 ± 1.10 for spoiled fish, with all ΔE > 12. The response sensitivity,
tervals and are represented by ΔE (Fig. 4a). The indicator was purple,
S (%), quantified using RGB analysis, increased from 32.21 ± 3.61
with ΔE value of 39.14 ± 1.74 and 44.44 ± 2.14 after 7 and 14 days of
(fresh), 79.09 ± 3.41 (spoiling) to 388.01 ± 0.59 (spoiled). The DFA
storage at room temperature (25 ◦ C), respectively, with good stability
indicator showed similar color changes when used to monitor spoilage
for approximately two weeks. The indicator was dark purple (ΔE > 50)
of mackerel fish at room temperature, in correlation with pH changes
after 14 days and turned green (58.48 ± 0.44) on the 35th day. The
(Table S4, Supporting Information). These findings indicate that the
change in color response might be attributed to the breakdown of an­
DFA indicator can differentiate fish quality both qualitatively and
thocyanins into relatively unstable aldehydes and phenolic acid

Table 3
Comparison of various anthocyanins (extracted from red cabbage) based fish freshness indicators.
Sl. Substrate for Steps involved in the preparation of the Image Color analysis Key features Year
No incorporating indicator capture Ref.
anthocyanins

1. Modified chitosan/ Solution preparation ~90 min – Colorimeter and The stability of anthocyanins in indicators was [35]
gelatin polyelectrolyte Solution casting and drying at 25 ◦ C for Photoshop software enhanced by the polyelectrolyte complex. The film
complex 48 h. was used as an indicator for fish spoilage.

2. Fish gelatin/Carbon Precursor solution preparation ~50 Smartphone Android application, The color changes were recorded using [78]
dots min SmartFood++ smartphones from different brands under diverse
Precursor solution is poured into the lighting conditions, resulting in the generation of a
petri dish and treated with 365 nm UV comprehensive dataset for the purpose of machine
light for 45 min, later placed in learning.
desiccator for 48 h.

3. Poly(ε-caprolactone) Solution preparation ~3h – Colorimeter The indicator changed from pink to purple in [32]
Electrospinning response to fish spoilage.

4. Basil seed gum/ Solution preparation ~2h Digital ImageJ The film showed a color change to green after fish [33]
chitosan Solution casting and drying at 25 ◦ C for camera spoilage.
48 h.

5. Gelatin/soybean Solution preparation ~2h – – The color of the indicator changed according to the [26]
polysaccharide/ Casting and drying for ~14 h degree of spoilage of the fish, exhibiting dark
phycocyanin orange, pink, and dark blue.

6. Carboxymethyl Solution preparation ~36 h Mobile ImageJ The color of the indicator changed from purple to [25]
chitosan/oxidized Screen-printing phone green according to the degree of spoilage of the
sodium alginate fish.

7. Jute fiber Delignification ~30 min Smartphone L*, a*, b* and RGB The degree of fish spoilage was reflected by the This
Dip coating ~10 min analysis android color changes observed in the indicator, which work
application ranged from pink, blue and green.

8
C. K P et al. Microchemical Journal 204 (2024) 111068

Fig. 4. The stability of the DFA indicator by its color response (ΔE) to pH 7.00 when stored at) room temperature and b) refrigerator, recorded at various
time intervals.

intermediates when stored at room temperature [79]. The DFA indicator & editing, Supervision.
exhibited a visible color change for approximately a month when stored
at room temperature, but its stability was improved when stored at 4 ◦ C.
Declaration of competing interest
The refrigerated indicator responded to pH changes and showed a
noticeable color change with a slight increase in ΔE for approximately
The authors declare that they have no known competing financial
75 days (Fig. 4b).
interests or personal relationships that could have appeared to influence
The DFA indicator showed remarkable stability when tested at 4 and
the work reported in this paper.
25 ◦ C due to the enhanced porosity and dye absorbability after
delignification. This implies that it can be an effective tool for moni­
Data availability
toring the spoilage of fish and other perishable foods, as perishable foods
have a shelf life of less than two weeks.
Data will be made available on request.
4. Conclusions
Acknowledgments
In summary, an easy-to-use, inexpensive, and non-toxic fish fresh­
Authors acknowledge St Joseph’s University, Bengaluru, Karnataka,
ness indicator (delignified jute fibers incorporated with anthocyanins,
India for TGA analysis. Chaithra K P acknowledges CHRIST (Deemed to
DFA) was fabricated using delignified jute (Corchorus olitorius) fiber as a
be University) for the research fellowship. Vinod T. P. is thankful to
substrate by incorporating halochromic anthocyanins from red cabbage,
Centre for Research Projects (CRP), CHRIST (Deemed to be University),
which show a visible color change in response to pH. The enhanced
for the Seed Money Grant SMSS-2334 and to Vision Group on Science
porosity after delignification, ability to adsorb dyes, and white color of
and Technology, Govt. of Karnataka for K-FIST-L1 grant (GRD No.
DF make it an ideal substrate for incorporating anthocyanins through
1143).
dip coating. When the indicator was exposed to various concentrations
of volatile amines, it showed a visible color change from pink to blue to
green and finally to yellow, depending on the concentration. The color Appendix A. Supplementary data
changes were captured using a smartphone, and a free application was
utilized for quantitative assessment using RGB and L*, a*, b* analyses, Supplementary data to this article can be found online at https://doi.
leading to the development of lab-free analytical technology. In addi­ org/10.1016/j.microc.2024.111068.
tion, when the indicator was used as a mackerel fish freshness indicator,
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