Tavuk etinde tazeliği izlemek için gerçek zamanlı-pH duyarlı kolorimetrik akıllı indikatör geliştirilmesi
Development of real time-pH-sensitive colorimetric intelligent indicator to monitor chicken meat freshness
Yazar: İREM MELDA KARACA
Danışman: PROF. DR. ZEHRA AYHAN
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2023
Konu: Gıda Mühendisliği = Food Engineering

Özet:
Bu tez kapsamında tavuk etinin tazeliğini gerçek zamanlı olarak izlemek ve bozulma seviyesini tespit etmek amacıyla renk değişimine bağlı akıllı tazelik indikatörü geliştirilmesi hedeflenmiştir. Tazelik indikatörleri tavuk etinin mikrobiyolojik olarak bozulması sonucunda meydana gelen CO2 metabolitine hassasiyet gösterecek şekilde 3 katmanlı olarak tasarlanmıştır. Tazelik indikatörlerinin renk değişim katmanında; %1 (a/h) oranında bromotimol mavisi (BTB) ve fenol kırmızısı (PR) boyaları, bağlayıcı olarak %3 oranında (a/h) metil selüloz (MC), plastikleştirici olarak %1 oranında (a/h) polietilen glikol (PEG-400) ve sodyum hidroksit (NaOH) doping solüsyonu (500µl), iç katmanda düşük yoğunluklu beyaz polietilen (LDPE) ve dış katmanda polietilen tereftalat (PET) filmleri kullanılmıştır. Katmanlar birbirine laminasyon işlemi ile yapıştırılmış ve 3 katmanlı kolorimetrik tazelik indikatörleri hazırlanmıştır. 
Geliştirilen tazelik indikatörlerinin CO2 metaboliti varlığında renk değişimlerini izlemek ve en iyi renk dönüşümünü sağlayan indikatörü tespit edebilmek amacıyla simülasyon çalışmaları gerçekleştirilmiştir. Simülasyon çalışmaları, gerçek gıda ambalajlama ve depolama koşullarını sağlamak amacıyla akıllı indikatör içeren PA/PE torba ambalajlara %0-30 CO2 enjekte edilerek ve 4℃'de 10 gün depolama yapılarak yürütülmüştür. Tazelik indikatörlerinin depolama süresince renk değişimleri, portatif bir kolorimetre ile L*, a*, b*, ∆E ve ImageJ yazılımı ile R, G, B, ∆RGB değerleri olmak üzere iki farklı renk ölçüm yöntemi ile tespit edilmiştir. Tazelik indikatörlerinin etkinliğini test etmek amacıyla gıda validasyonu çalışması yapılmış olup bu amaçla tavuk göğüs etleri, poliamid/polietilen (PA/PE) torba ambalajlarda hava atmosferi (%80 N2 ve %20 O2) ve %100 N2 atmosferi altında ambalajlanmış ve 4℃'de 10 gün süreyle depolanmıştır. Validasyon çalışmasında depolama süresince tavuk göğüs etinin kalitesindeki değişimler ve bozulma süreçleri tepe boşluğu gaz kompozisyonu, pH, toplam uçucu bazik nitrojen (TVB-N), trimetilamin (TMA), toplam mezofilik aerobik bakteri, Pseuomonas spp. ve duyusal değerlendirme analizleri ile izlenmiştir. Tazelik indikatörlerinin depolama süresince renk değişimleri L*, a*, b*, ∆E ve R, G, B, ∆RGB değerleri ile belirlenmiştir. 
Simülasyon çalışmasının ön deneme sonuçlarına göre en belirgin renk dönüşümü %1 pH boyası içeren dopingli indikatörlerde gözlenmiştir, bu sonuçlara istinaden ana simülasyon denemesi ve gıda validasyonu  çalışması bu indikatörler ile yürütülmüştür. Simülasyon çalışmasının ana denemelerinde, BTB ve PR bazlı kolorimetrik tazelik indikatörlerinde CO2 konsantrasyonundaki artış ile kademeli bir renk değişimi gözlenmiştir. BTB bazlı tazelik indikatörlerinde %10-15 CO2 konsantrasyonlarında turkuazdan koyu yeşile, %15-30 CO2 aralığında koyu yeşilden açık yeşile kademeli bir renk değişimi gözlenmiştir. PR bazlı indikatörlerde %5 CO2 konsantrasyonunda mordan kırmızıya, %5-30 CO2 aralığında ise turuncu ve sarımsı-turuncuya renk değişimi gözlenmiştir. 
Gıda validasyon çalışmasında, 4℃'de 10 gün süreyle ambalajlanan tavuk göğüs etlerinin raf ömrü hava atmosferi ile ambalajlanan grupta 4 gün, %100 N2 atmosferi ile ambalajlanan grupta ise 6 gün olarak belirlenmiştir. PR bazlı indikatörlerin rengi her iki atmosferde de mor ile koyu kırmızı arasında değişmiş olmasına rağmen depolama süresince artan CO2 konsantrasyonu ve diğer kalite parametreleri ile belirgin bir korelasyon göstermemiştir. Her iki atmosferde de PR bazlı tazelik indikatörlerinin toplam renk değişimi değerleri artmasına rağmen bozulma sınırı olarak belirtilen %10-15 CO2 aralığında tüketici tarafından çıplak gözle ayırt edilebilecek belirgin bir renk geçişi tespit edilmemiştir. Depolamanın başlangıcında koyu mavi olan BTB bazlı indikatörler, depolamanın 2. ve 4. günlerde turkuaz, depolamanın 6. gününde ise koyu yeşil olarak gözlenmiştir. BTB bazlı indikatörde gözlenen bu renk değişimi, diğer kalite parametreleri ve toplam renk değişimi değerleri ile ilişkili bulunmuştur. % 100 N2 atmosferi ambalajında depolamanın 8. gününde %7,03 olarak belirlenen CO2 konsantrasyonunda BTB bazlı indikatörler turkuazdan biraz daha belirgin bir yeşile renk dönüşümü göstermiştir. BTB bazlı indikatörlerin simülasyon çalışmalarında depolamanın 4. ve 6. günlerinde %10-15 CO2 konsantrasyonlarındaki turkuazdan koyu yeşile renk değişimi, gıda validasyonunda hava atmosferi altında ambalajlanan indikatörler ile oldukça benzer bulunmuştur. Ancak her iki atmosfer için de PR bazlı indikatörlerdeki renk değişimleri, simülasyon çalışmalarındaki kadar belirgin bulunmamıştır. 
Genel olarak değerlendirildiğinde, BTB bazlı tazelik indikatörlerinde depolama süresince koyu mavi, turkuaz ve koyu yeşil olarak gözlenen 3 aşamalı renk değişimi, tüketicilere "taze, hala taze ve bozulmuş" sinyallerini vererek gıdadaki kalite değişimlerinin gözle görülür şekilde izlenmesini sağlayabilecektir. BTB bazlı tazelik indikatörleri, tavuk ve kanatlı etlerinde gıda kalitesinin gerçek zamanlı olarak belirlenmesinde ve gıda güvenliğinin sağlanmasında endüstriyel ölçekte umut verici bulunmuştur.

Abstract:
In this thesis, an intelligent freshness indicator based on color change was developed to monitor the freshness/spoilage of chicken meat in real-time. The freshness indicators were designed in three layers to be sensitive to the CO2 metabolite occured due to microbiological spoilage of chicken meat. In the color change layer of the freshness indicators, bromothymol blue (BTB) and phenol red (PR) dyes were used at a ratio of 1% (w/w), along with a 3% (w/w) methyl cellulose as a binder, 1% (w/w) polyethylene glycol (PEG-400) as a plasticizer, and a doping solution of sodium hydroxide (NaOH) (500µl). The freshness indicators were laminated with low-density white polyethylene (LDPE) used in the inner layer and polyethylene terephthalate (PET) films in the outer layer.
Simulation studies were performed to monitor the color changes of the freshness indicators in the presence of metabolites and determine the best indicator formulation by simulating CO2 concentrations ranging from 0% to 30%. The simulation studies were conducted at 4℃ for 10 days to provide real food packaging and storage conditions. The color changes of the freshness indicators images were recorded with a portable scanner and mobile phone camera. The total color change values were determined using two different color measurement methods. L*, a*, and b* values were measured by using a colorimeter method and the total color difference (∆E) was calculated. Image J software was used to evaluate the color R, G, B, and total color difference (∆RGB) values were calculated by loading the recorded digital images into Image J software.
To test the functionality of the freshness indicators, a food validation study was conducted at 4℃ for 10 day using chicken breast meat, which was packaged in polyamide/polyethylene (PA/PE) with an air (80% N2 and 20% O2) and a 100% N2 atmospheres. During the validation study, changes in the quality parameters of the chicken breast meat were monitored using headspace gas composition, pH, total volatile basic nitrogen (TVB-N), trimethylamine (TMA) concentration with gas chromatography (GC), total mesophilic aerobic bacteria, Pseudomonas spp., and sensory evaluation. The color changes of the freshness indicators during storage were determined using a portable colorimeter with L*, a*, b*, ∆E, and ImageJ software using R, G, B, and ∆RGB values.
According to the pre-simulation results, the most significant color change was observed in colorimetric freshness indicators containing 1% pH dye and doping solution. Therefore, the main simulation and food validation studies were conducted using these freshness indicators. In the main simulation study, a gradual color change was observed with increasing CO2 concentration in both BTB-based and PR-based colorimetric freshness indicators.
In the case of BTB-based freshness indicators, a gradual color change was observed from turquoise to dark green at CO2 concentrations of 10-15%, and from dark green to light green in the range of 15-30% CO2. For PR-based indicators, a color change from purple to red was observed at a 5% CO2 concentration, and from orange to yellowish-orange in the 5-30% CO2 range. The ∆E and ∆RGB values for both indicators were found to be associated with the indicator images showing color change as a result of increasing CO2 concentration.
The % O2 concentration decreased significantly during storage in the air atmosphere, while it remained stable and low in the 100% N2 atmosphere (p≤0.05). The significant increase in % CO2 concentration was determined in both atmospheres (p≤0.05). The CO2 concentration in the air atmosphere increased gradually, reaching 12.89% on the 6th day of storage. The CO2 concentration in the 100% N2 atmosphere reached 8.98% at the end of the storage. CO2 concentration is considered a good indicator of spoilage, as it correlates well with microbial growth.
The pH of fresh chicken breast was 6.12 at the beginning of storage and decreased gradually in both air and 100% N2 atmospheres. In the air atmosphere, pH of the chicken decreased to 5.86 on the 4th day of storage and then increased to 6.20 on the 8th day (p ≤ 0.05). Similarly, in the 100% N2, pH decreased to 5.93 on the 6th day and then increased to 6.04 on the 10th day. The initial pH reduction was attributed to lactic acid formation by microbial growth, while the increase in pH afterwards was likely due to proteolytic enzyme activities of the microbial flora. These enzymes break down proteins and can lead to unpleasant changes in sensory properties of the chicken breast.
The TVB-N values of chicken breasts gradually and significantly increased during storage in both air and 100% N2 atmospheres (p≤0.05). TVB-N values were higher in the air atmosphere compared to the 100% N2 atmosphere. While the initial concentration of TVB-N was 7.67 mg/100 g in the fresh product (day 0) under air atmosphere, it was determined as 19.44 mg/100 g on the 4th day and 28.72 mg/ 100 g on the 6th day of storage. Similarly, TVB-N increased to 11.51 mg/100 g on the 4th day of storage and determined as 19.35 mg/100 g on the 6th day and reached to 21.90 mg/100 g on the 8th day under 100% N2 atmosphere. TVB-N is an important metabolite correlated with spoilage of chicken breast meat, and an increase in TVB-N values indicates a reduction in freshness of the chicken meat.
The TMA concentrations of chicken breasts under air and 100% N2 atmospheres were measured and compared to the upper limit of acceptance suggested for fresh chicken meat. The TMA concentrations gradually increased during storage, but the increments were not statistically significant (p>0.05). The TMA concentration in air atmosphere reached the upper limit of acceptance (2.5 and 2.6 mg N/100 g) on the 6th and 8th day of storage, while for 100% N2 atmosphere, the TMA concentrations remained below the limit.
Both TMAB and Pseudomonas spp. levels significantly increased during storage under both atmospheres (p≤0.05). The initial TMAB and Pseudomonas spp. levels were 3.38 and 3.62 log cfu/g, respectively. The TMAB load reached 5.10 and 6.64 log cfu/g, while the Pseudomonas spp. level reached 6.21 and 7.40 log cfu/g on the 4th and 6th day of storage under air atmosphere, respectively. In the 100% N2 atmosphere, the TMAB level was 6.16 and 6.62 log cfu/g, while the Pseudomonas spp. level was 5.96 and 6.88 log cfu/g on the 6th and 8th day of storage, respectively. 
The sensory quality of the chicken breast changed significantly during storage in both air and 100% N2 atmospheres (p≤0.05). Appearance, odor, texture, and overall acceptance decreased below the acceptable limit on the 6th day of storage in the air atmosphere, while they remained above the limit in the 100% N2 atmosphere. The shelf life of the air and 100% N2 packed chicken breast meats was determined as 4 and 6 days, respectively, based on the CO2, TVB-N, microbiological, and sensory evaluation. Sensory evaluation scores remained below 3 points after the 4th day in the air atmosphere and the 6th day in the 100% N2 atmosphere.
The color change of two types of freshness indicators (PR-based and BTB-based) were evaluated during the storage of chicken breast under two different atmospheres (air and 100% N2). The results showed that both types of indicators had an increase in color difference (represented by ∆E and ∆RGB) with an increase in CO2 concentration under both atmospheres. For PR-based indicators, the ∆E and ∆RGB values increased gradually up to 25.26 and 87.35, respectively, in air atmosphere and up to 20.71 and 56.66, respectively, in 100% N2. Meanwhile, BTB-based indicators showed a higher total color difference during the storage period for both atmospheres. The ∆E and ∆RGB value of the BTB-based indicators increased up to 39.95 and 115.86, respectively, in air atmosphere, and up to 32.97 and 89.09, respectively, in 100% N2 atmosphere. The PR-based indicators changed from purple to dark red on the 2nd day of storage and then to light red-orange on the 6th and 8th days, eventually turning reddish-orange on the 10th day. The BTB-based indicators started as dark blue and turned turquoise on the 2nd day, then green and dark green on the 4th day in both air and 100% N2. On the 6th day of air atmosphere, the color turned to dark and light green, and on the 8th day of 100% N2 atmosphere, the color became lighter green. On the 10th day, the BTB-based indicators turned to a light green color with yellow surroundings in both atmospheric conditions.
The total color differences of BTB-based indicators are in a very good correlation with changes in CO2 concentration and showed a better correlation than PR-based indicators. The increase in CO2 concentration for both atmospheres during the storage period did not have much effect on the PR-based indicator visuals. The color of the PR- based indicators changed between the purple and dark red, during storage but color transition was not clear during the shelf life, in both of air and 100% N2 atmospheres. During the storage, visual color changes of the indicators have shown that BTB-based indicators are in a well correlation with changes in quality parameter changes and reflect the freshness level of chicken breast meat simultaneously, in both atmospheres. During the simulation studies, a noticeable color change with naked eye from turquoise to dark green was observed for BTB-based indicators when the CO2 concentrations reached 10-15% on the 4th and 6th days of storage. Similar color changes were also observed in the indicators packaged under air atmosphere during the subsequent food validation. This indicates a close resemblance between the color change observed in the simulation and food validation studies. Conversely, the color changes observed in PR-based indicators for both atmospheric conditions in food validation were not as pronounced as those observed in the simulation studies. Specifically, the distinct orange-yellow color observed at a CO2 concentration of 10-15% in the simulation study were not detected during the food validation study.
The BTB-based freshness indicators showed a clear, 3-stage color change during the 6 days of storage under air atmosphere. The colors were dark blue (0-2 days), turquoise (2-4 days), and light green (after 6 days) to signal "fresh, still fresh, and spoiled". A similar color change trend was observed in 100% N2 atmosphere, with the most significant color change observed between the 8th and 10th days. The color changes under the 100% N2 were dark blue (0th day), turquoise close to blue (2nd day), turquoise (4th day), greenish-turquoise (6th day), dark green (8th day), and dark green (10th day). The BTB-based freshness indicators gave a three-stage response to spoilage under both air and 100% N2 atmospheres while the PR-based freshness indicators did not provide clear color changes for consumers to distinguish spoilage limits with the naked eye.
In conclusion, BTB-based three-layer colorimetrik pH-sensitive freshness indicators are more effective in detecting CO2 metabolite in chicken meat and have a higher potential for use in food packaging. The three-stage color changes observed in the indicators correlated well with spoilage especially considering microbiological and sensory quality. The study also suggested the shelf life of chicken breast meat as 4 days under air atmosphere and 6 days under 100%N2. BTB-based colorimetric freshness indicators were found to have the potential to be adapted to industrial scale to monitor the freshness/spoilage of chicken meat in real time. The use of freshness indicators is promising for producers and consumers, as it ensures food safety, reduces waste, and promotes traceability and sustainability in the food industry.