Number of Microorganisms and Level of Spoilage Relationship

1.0 Materials and Method
1.1 Chemicals
50 g of dried powder of curcumin will be extracted in a Soxhlet apparatus with 500 mL of 95% ethanol. The process will be done until the solvent is colourless. The extract then will be filtered and concentrated using a rotary evaporator. The dried ethanolic extract will be stored for further usage. A stock solution of curcumin will be prepared by dissolving 10 mg of dried ethanolic extract in 10mL of ethanol (50%) to give a concentration of 1 mg/mL. All chemicals that will be used are reagent grade (will be supplied by Merck, Sigma, of Fluka) and will be used as supplied.
1.2 Methods
1.2.1 Preparation of Bacterial Cellulose Membrane
Acetobacter xylinum culture will be cultivated using a Herstin-Schramm nutrient (HS) medium that consists of glucose (2 w/v%), yeast extract (0.5 w/v%), bacto-pepton (0.5 w/v%), citric acid (0.115 w/v%), Na2HPO4 (0.27 w/v%), MgSO4_7H2O (0.05 w/v%) and ethanol (1 v%) which will be added after the base has been sterilised. The culture will be cultivated in stationary conditions.
The HS medium will be filled in 300 cm3 conic flasks. The bacterial breeding process will be conducted in a period of seven days at 30oC, which inoculums will be grafted at approximately 4 w% in relation to the HS medium. Glucose, arabinose, mannose, galactose, xylose and mannitol will be used as carbon sources in the biosynthesis process of bacterial cellulose. The membrane of the bacterial cellulose will be treated with NaOH (approximately 5% concentration) for 60 minutes and a temperature of 100oC so that bacterial cells can be removed and substrate from the inner layers of the bacterial cellulose film. The bacterial cells will then be rinsed with tap water until it achieves a neutral condition at around pH 7.0.
To prepare the membrane sheet, 10 g of the bacterial cellulose will be blended until it is homogenous. Then, it will be casted onto the glass plate and pressed. The membrane is to be left overnight for 12 hours, and will be dried at 60 oC. The membrane sheet is to be stored away.
1.2.2 Immobilization of Natural Dye on Bacterial Cellulose Membrane
The natural dye will be immobilized onto the bacterial cellulose membrane using absorption method. The membrane sheet will be immersed into 10 mL of curcumin stock solution (1 mg/mL) at ambient temperature for 12 hours. Then, the curcumin/cellulose membrane will be rinsed with tap water to ensure that unbound indicator within the membrane will be removed. The curcumin/cellulose membrane will be dried using an electrical drier. Finally, the curcumin/cellulose membrane will be cut into shapes according to the design of on-package sticker sensor (Figure 1).

Figure 1:The design of the sticker sensor based on curcumin/cellulose membrane for broiler chicken cuts’ freshness with colour indication for fresh, medium (need to be consumed in hours) and not fresh (spoilage, do not consume).
1.2.3 Preparation of Broiler Chicken Cut Samples
In this study, a fresh boiler chicken of normal pH (5.5-5.6) will be used. The chicken will be cut into a 100 g and 50 g portions for microbiological and sensory analysis respectively. Each portion will be placed in a low-density polyethylene plastic film (0.9 g/cm3) which will be put on plastic trays. Then, the samples will be stored in a low-temperature incubator (4-0.2 oC) and in room temperature. The temperature of the samples will be observed throughout the entire storage period with electronic temperature recording devices. Four sample packages of the chicken cut product will be taken at appropriate time samples from each storage temperature. They will be analysed for microbial growth, pH and sensory characteristics such as colour and odour. The test will be repeated for three times for each sample to increase the accuracy of the result. This means that 12 determinations in total will be taken per test condition. Then, the average value of the three determinations will be used per sample for the statistical analysis.
1.2.4 Microbiological Analysis
Samples of chicken cuts will be weighed for 25 g each. Then, they will be added to quarter strength Ringer’s solution (225 mL) and will be homogenized in a stomacher for 60 seconds at room temperature. Decimal serial dilutions in quarter strength Ringer’s solution will then be prepared, 1 mL of 0.1 mL samples of appropriate dilutions will be spread on the surface of appropriate media in petri dishes to count (a) total aerobic viable count (TVC) on plate count agar; incubated at 25oC for 72 hours, and (b) Pseudomonas spp. on cetrimide-fucidin-cephaloridine (CFC) agar; incubated at 25oC for 48 hours. Both plates will be examined to observe typical colony types and morphological characteristics that were associated with each growth medium. Furthermore, the selectivity of each medium will be monitored regularly by Gram staining. Smears that will be prepared from randomly selected colonies from both media will also be examined using microscopic examination
1.2.5 Measurement of pH and VA in Broiler Chicken Cut Samples
The glass electrode of a pH meter will be immersed in the homogenate of chicken meat to record the pH values at the end of microbiological analysis. Perchloric acid extracts will be taken from the chicken meat samples for analysis of TVBN levels. All the chicken meat samples will be rinsed thoroughly with tap water. The chicken meat will then skinned and minced through a meat grinder with 4 mm holes, three times. 10 g of chicken meat sample will be blended with 90 mL of PCA 6%. 50 mL of the filtrate is to be made alkaline using hydroxide 20% and distilled water for a period of 10 minutes in a 2100 Kjeltec Distillation Unit. The process will be repeated three times.
1.2.6 Sensory Analysis
Sensory evaluation of chicken cut samples will be performed by a five-member (staff from the laboratory) sensory panel. It will be performed during storage to both chicken cut samples under chiller and room conditions. The same persons will be used in each evaluation session, but they will not know the age and temperature history of the product being tested. The evaluation will be carried out under artificial light, and the temperature of the product will be of the ambient temperature. When evaluating the product, special attention will be given to the colour, texture and odour of the chicken meat. The texture of the chicken meat will be measured by a texture meter while the odour will be judged and recorded in appropriate forms with descriptive terms, showing the organoleptic evolution of quality deterioration. A simple three-point scoring system will be used. Each characteristic will be scored on a continuous 0 to 3 hedonic scale, with 0 being the highest quality score, 1 for the acceptable product, 2 as the limit of product acceptance or rejection point and 3 is the unacceptable chicken cut sample.
1.2.7 Measurement of the Visual Sticker Sensor Response
The sticker sensor is made of natural dye of curcumin immobilised on bacterial cellulose and designed as in Figure 1. The sticker sensor will be placed on the packaging of the chicken cut samples, with direct contact to the atmosphere in the package through a hole that attached to the sensor. Then, the chicken cut packages will be stored at chiller and room temperature in order to assess the applicability of the developed sticker sensor to observe the spoilage process of the product. The irreversible colour change of the sensor from the initial yellow to reddish orange will be used as the measurable response of change. The kinetics of colour change of the sticker sensor will be evaluated by a hand-held colorimeter to determine the CIE colour space coordinates.
2.0 Expected Results
The objectives of this study are to investigate the relationship between the number of microorganisms and level of spoilage, and also to develop an indicator to monitor the freshness of chicken. A few tests will be carried out, including microbiological analysis, pH and TVBN analysis, sensory analysis, as well as the response of the developed sticker sensor. The expected results for this experiment are:

When the number of microorganism increase, the level of spoilage is higher.
The pH and TVBN levels increase when the level of spoilage is higher.
The sensory score will increase as the level of spoilage increase.
The developed sticker sensor will remain the original colour yellow when the chicken cuts is fresh, orange when the product should be consumed in a few hours, reddish orange when the product is spoiled.

3.0 Abstract
A few studies have shown that as the level of microbial growth increase, the level of spoilage also increase. The main objective of this study is to develop a sensor to indicate the level of spoilage that can be seen using our naked eye. The smart packaging that was invented really helped the consumers to judge the freshness of raw materials. There are a few indicator of meat freshness that has been studied, such as colour-based pH indicators and volatile compounds indicator. Examples of indicators are methyl red, natural dye of curcumin and colorimetric sensors array using e-nose. A sticker sensor will be developed using natural dye curcumin immobilized on bacterial cellulose. A few tests will be done, that are microbiological analysis, pH and TVBN analysis, sensory analysis, and the response of the developed sticker sensor to meet the goals of this study. The expected result will be the number of microorganism, pH and TVBN levels and sensory score will increase when the level of spoilage increase. The sticker sensor will turn colour from yellow, to orange, and finally reddish orange indicating fresh, not really fresh and spoiled respectively.