The document discusses developing pH-sensitive smart packaging films using natural food colorants like anthocyanins extracted from blueberry peels to monitor the quality and safety of fish products. The films are designed to change color based on pH changes during fish spoilage caused by microbial growth and the production of volatile nitrogen compounds. Testing of the films packaged with fish showed they transitioned from reddish to light pink after 72 hours of storage, corresponding to a pH increase indicating spoilage. The films have potential for use as smart indicators in food packaging to help reduce waste by allowing consumers to easily assess food freshness without opening packages.

1. pH-SENSITIVE (HALOCHROMIC) SMART PACKAGING FILMS
BASED ON NATURAL FOOD COLORANTS FOR THE
MONITORING OF QUALITY AND SAFETY OF FISH PRODUCTS
Prepared by
NIMAH SALALH ALNEMARI (GS55972)
Date: th
July 2021
Introduction
Consumers are getting more demanding for fresh fish high in quality to ensure proper nutrition
and a healthy diet. Fish products are major sources of complete proteins and play important role
in the human diet including provision of omega-3 among a variety of health benefits. However,
they are highly perishable due to enzymatic reactions and microbial contamination (Zhang et al.,
2019). The development of improved methods to determine food quality such as freshness,
microbial spoilage, oxidative rancidity or oxygen and/or heat-induced deterioration is extremely
important to food manufacturers. To maximize the quality and safety of foodstuffs, prediction of
shelf-life based on standard quality control procedures is normally undertaken. Intelligent
packaging, as a new technology, has been recently offered to inform real-time freshness and

2. monitor critical factors affecting food quality (Mohan, 2019). One mean of doing so is through
intelligent packaging incorporating gas sensor technology for sensing oxygen and CO2.
The monitoring of these gases in the package helps in maintaining the food quality (Remya et al.,
2017). Gas chromatography-mass spectrometry (GC/MS) is one of the common analytical
instrumental techniques available to monitor gas compositions in modified atmosphere packaged
(MAP) products. The technique requires breakage of package integrity and is time-consuming and
expensive. Portable headspace oxygen and/or carbon dioxide gas analysers use ‘minimally
destructive’ techniques (packages can be resealed) but tend not to apply to real-time, online control
of packaging processes or large scale usage.
An optical sensor approach offers a realistic alternative to such conventional methods. It can be
used as a leak indicator or to verify the efficiency of O2 scavenger, CO2 emitter or MAP systems
(Vermeiren et al., 1999). Films containing colorimetric indicators that are sensitive to changes in
pH and/or chemical composition are useful in the development of smart packaging materials for
fresh foods (Alizadeh-Sani et al., 2020a; Alizadeh-Sani et al., 2020b). The indicators use
commonly assume a colour change as a result of a chemical or enzymatic reaction. Considering
pH changes, which reveal great importance to notify spoilage, development of visible pH-sensitive
indicators (e.g. intelligent packaging system) has gradually expanded to recognize fish spoilage
stages. Hence, there is great potential in creating a new generation of smart and active
biodegradable films for food applications, with the ultimate aim of increasing food safety, quality,
and sustainability. To achieve the aim, many are interested in identifying natural substances that
can be used as indicators, antimicrobials and/or antioxidants with biodegradable films.

3. Chitin nanofibers (ChNFs) can be isolated and used as sustainable O2 barrier materials for food
packaging. These bioavailable nanomaterials are readily dispersed in water enabling spray-coated
films to be deposited at high rates onto uneven or delicate surfaces. ChNFs also exhibit a high
modulus and tensile strength and are excellent candidates for gas barrier films (Wu, 2014). In a
recent study, ChNF/CNC multilayers lead to a reduction in the O2 permeability of the final
composite film by as much as 73%. The nanocrystalline or nanofibrous forms of these biopolymers
represent promising renewable, compostable sources of materials for replacing traditional plastics
in packaging applications. Figure 1 shows the biodegradable films with two alternating coated
layers consisting of poly(lactic acid) PLA-(CNC-ChNF), which can reduce O2 permeability relative
to neat PLA, even at elevated relative humidity (RH) of 70%. Interestingly, these improvements
in O2 barrier properties were not observed for films with single layers of neat ChNF or CNC
sprayed from an equivalent volume. (Ifuku & Saitomo, 2012)
Figure:1 ChNF/CNC multilayers were found to lead to a reduction in the O2 permeability of the final
composite film
(Satam, 2018)
Blueberry peels are a byproduct of blueberry juice processing and contain higher quantities of
anthocyanins (primarily in their glycosylated forms) than those of other fruits and vegetables
(Giovanelli & Buratti, 2009). The colors of anthocyanins extracted from blueberry peels vary

4. widely and correspond to different pH values. Moreover, the low cost, safety and wide color range
of anthocyanins extracted from blueberry peels make them attractive for use in intelligent
packaging production. Overall, biodegradable materials are receiving more attention in packaging
applications because of their safety and unique degradation properties (Tan et al., 2013).
Active packaging using pH-sensitive (halochromic) films for shelf life prediction of fish
The spoilage of seafood occurs primarily as a result of the action of microorganisms and enzymes,
which induce chemical changes in the seafood. Fish spoilage leads to the generation of volatile
basic nitrogen compounds from microbial processes, which causes the pH of the fish to increase.
Total volatile base nitrogen (TVB-N), K-value and total aerobic counts (TACs) are of importance
in freshness evaluation. In this light, the colorimetric pH-sensing film provided significant
antioxidant and antimicrobial activity. Using fish as an example, as in situ colorimetric indicators
of food freshness/spoilage. The typical experimental setup for testing of pH-sensing film with fish
is shown in Figure 2.
Figure 2: Experimental design of pH indicator for detection of fish freshness.
(Rastiani, 2019):

5. Changes in the environment are the main cause of the formation of volatile nitrogen compounds
and an increase in the pH value. These volatile compounds include ammonia and amines, which
are formed as proteins degrade. Thus, red barberry anthocyanin (RBA)-loaded CNF films may be
used as smart indicators of fish freshness after 72 hours of storage at room temperature (Alizadeh-
Sani et al. 2020b). During the test period (72 h), the pH values of the fish sample increased from
6.3 to 8.0. The resulting rise in pH within the films resulted in a visible color transition, with the
films going from reddish to light pink (Figure 3). The findings are consistent with those of other
researchers who have published on the use of natural pigments in biopolymer films to track food
safety and quality (Tavassoli et al., 2020; Huang et al., 2019; Kang et al., 2020).
Figure 3: Monitoring the freshness and spoilage of fish fillets using smart halochromic film.

6. (Sani, 2021)
It has been reported that during the spoilage process, low molecular weight substances and alkaline
compounds particularly, ammonia and trimethylamine, which are generally known as TVB-N, are
produced and correspond for sensorial rejection and off-flavors. TVB-N is a distinctive index to
assess fish spoilage. Samples with TVB-N higher than 25 mg/l00 g are considered spoiled food
(Dalgaard et al., 2006, Ghaly et al., 2010). The initial TVB-N value of fish was 13.53 mg/100 g,
indicating the freshness of samples. TVB-N subsequently increased accompanied by TPC and pH
during storage at 4 °C. The increasing trend of TVB-N value can be attributed to protein
degradation by microbial activity and autolytic enzymes resulting in deamination of amino acids
and accumulation of volatile base. Figure 4 shows the color changes of the pH-sensitive film as
the TVB-N increases.

7. Figure 4: The sensitivity of smart halochromic indicator versus ammonia vapor.
(Sani, 2021 )
Application of smart packaging using natural food colorants-based pH-sensitive
(halochromic) film with for the monitoring of quality and safety of the fish
Without opening the package, customers can easily differentiate between fresh and expired fish
through the freshness indication showed by the pH-sensitive film, which may lead to
improvements in food quality and reductions in food waste (Yam et al., 2005). Moreover, a smart
indicator embedded within the final packaging material can be used to monitor and record food
status throughout storage by coupling it with analytical sensors (Ghaani et al., 2016;
Mohammadian, Alizadeh-Sani, & Jafari, 2020). In addition, smart biodegradable films can be

8. further engineered to improve their functional performance by including antimicrobials and/or
antioxidants to protect a packaged food from spoilage during storage (Bagheri et al., 2019;
Alizadeh-Sani et al., 2020a).
Spoilage microorganisms can easily attack perishable fish and impose an adverse effect on the
quality and shelf life of postharvest fish products. In an evaluation of the microbial inhibitory of
the films, CNF and RBA both possess antimicrobial activity against foodborne pathogenic and
spoilage organisms such as Escherichia coli and Staphylococcus aureus (Alizadeh-Sani et al.
2020b). The addition of RBAs enhanced the films' inhibitory activity, which is usually due to
anthocyanins' polyphenolic structure (Zhang et al., 2019). Although the mechanism of action of
CNFs has not been fully elucidated, previous research indicates that it can be used as a
bacteriostatic agent. Indeed, CNF has been demonstrated to promote bacteria cell flocculation and
inhibit bacteria growth, presumably through chemical interactions between chitin (a positively
charged molecule) and cell membranes (negatively charged). These interactions can result in
bridging flocculation, resulting in membrane destruction and intracellular material leakage
(Shankar et al., 2015).
The RBA-loaded CNF films have high radical scavenging activity (82.1%) (Alizadeh-Sani et al.
2020b). This finding can be attributed to the high antioxidant activity of the RBA anthocyanins'
OH classes. Earl studies have also identified an increase in antioxidant activity following the
introduction of anthocyanins into packaging films (Huang et al., 2019; Wang et al., 2019; Yong et
al., 2019).

9. Conclusion
In order to effectively preserve the storage quality and extend the shelf-life of fresh fish, natural
pigments such as naphtoquinones and anthocyanins could be a viable alternative to synthetic dyes
for the production of smart packaging materials equipped with sensors/indicators capable of
continuously monitoring the freshness and spoilage of food products. They achieve these functions
by exhibiting quantifiable changes in their optical properties in response to changes in food
properties. These innovative packaging materials can increase food quality and safety, as well as
decrease food waste, thereby improving the food supply's healthiness and sustainability.
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