1

CHAPTER I INTRODUCTION

Background of Study Pork and fish, being diverse, highly nutritional, and widely distributed foods, are undoubtedly the most consumed meat products by the people. However, these are protein-rich foods which make them highly perishable and susceptible to spoilage caused by the presence of millions of bacteria which give off a rancid odor. The foul odors stem from the release of by products that occur when bacterium secretes protease, an enzyme that allows it to convert protein into amino acids, which then initiates the chemical breakdown or digestion of tissue (Kwasiborksi et al., 2008). In association with food spoilage comes food freshness. This is the condition of a food being new, usually raw, low in bacterial count and free from biogenic amines (Funazaki et al., 1995). It is affected by many factors such as pH, temperature, and amount of biogenic amines. It is the key characteristic of overall food quality and is the result of all the desirable characteristics that make food acceptable to eat. Although pork and fish products on the market carried statements on their packages, indicating its freshness for weeks from the purchase date;

2
chemical and biological tests may prove otherwise. By the time a consumers nose detects a rotten smell, spoilage bacteria have already grown from a few early inhabitants on a meat to a full blown contamination of a million or more colony forming units of bacteria per gram. Such large numbers of bacteria could pose immediate health risks which may even be fatal. Consequently, a simple test, known as bioassay, could detect the existence of multifold bacteria in foods. Such a test would provide a real measurement of freshness or wholesomeness; determine whether the pork or fish has begun to spoil even before the onslaught of disgusting and offensive odors, and even help prevent food-borne illnesses. This bioassay which would offer the vendor/s an easy-to-comprehend visual signal for freshness can be incorporated with bioluminescence. A bioluminescencebased bioassay can be used not only for the detection of products freshness but also for the presence of extensive contamination since bioluminescence is an oxygen requiring process that is also sensitive to many chemical contaminants. Meat undergoing spoilage because of the unusually high bacterial load should consume or deplete the surrounding oxygen rapidly and should likewise quickly shut off or inhibit bioluminescence. This gave the idea to the researchers to apply and develop a simple, rapid, inexpensive yet effective method of determining freshness of protein

3
rich foods like pork and fish fillet. Bioluminescence as a biological-sensing device would ensure that the public would obtain the freshest possible red and white meat product in wet markets.

Statement of the Problem Meat and sea foods that are sold in the market claims freshness or wholesomeness, however consumers have no way of knowing it for sure and examination of these foods based on organoleptic properties is quite subjective. Thus, this study will determine the effectiveness of a bioluminescence-based assay called Tube Bioluminescence Extinction Technology or TuBET in determining the freshness of ground pork and fish fillet based on their bacterial density.

Objectives of the Study Specifically, this study aims to: 1. Determine the increase in bacterial cell count of room temperature incubated fresh ground pork and fish fillet using the Breeds Method. 2. Determine the relationship of the direct bacterial count from the Breeds Method with the time of bioluminescence extinction in test tubes. 3. Determine the change in pH of room temperature incubated ground pork and fish fillet over a period of time.

4
4. Compare the sensitivity and assay period of TuBET with other existing chemical and microbiological methods of determining Freshness.

Significance of the Study Consumption of meats that are no longer fresh can damage the health and cause illness to the consumers. As such, there is a need for a method that can determine the Freshness of foods rapidly in the market to protect the Public Health. Bioluminescence-based methods can be the basis of a Green Microbiology or Green Chemistry exemplifying a simple, rapid, small carbon footprint and non-toxic method of determining bacterial count in fresh foods like pork and fish.

Scope and Limitations This study will use of the bioluminescence exhibited by Vibrio fischeri for TuBET. The samples whose freshness or wholesomeness were evaluated were ground pork and fish fillet from Decapterus macrosoma, or commonly known as Galunggong procured in wet markets. Direct cell counting using Breeds Method was used instead of the Standard Viable Plate Count Method because of the sheer numbers of sampling and replications required of the bioassay. The researchers made use of a 24- hour, room temperature incubation period averaging 30C. Statistical methods employed because of its analytical nature were the

5
mean, correlation coefficient and regression equation or least squares method.

CHAPTER II REVIEW OF RELATED LITERATURES

Bioluminescence Bioluminescence is a metabolic activity that requires oxygen to produce visible light. Its reaction is closely associated to the electron transport system of the marine luminous bacteria (Nealson & Hastings, 1992).The use of bioluminescent bacteria as biological indicators dates back in 1950s. Applications range from the assessment and monitoring of the toxicity of compounds to the monitoring of genetically engineered organisms released into the environment (Scheerer et al., 2006). One of the major requirements for bioluminescence is oxygen. The role of oxygen in bacteriafish bioluminescent associations is based primarily on experiments with luminous bacteria grown in laboratory culture. Such studies have shown that reduced oxygen availability increases the specific activity of bacterial light emission and depresses bacterial growth rate. These results have led to the hypothesis that the host might increase the efficiency of its metabolic investment in the association by controlling oxygen concentration (Ruby & McFall-Ngai, 1999).

7
Bioluminescence has emerged as a powerful method to analyze infectious diseases in animals, known as Bioluminescence imaging (BLI). BLI offers real-time monitoring of spatial and temporal progression of infection, as opposed to euthanizing a group of animals and quantifying colony or plaque forming units at multiple time points (Hutchens & Luker, 2007). Vibrio fischeri Vibrio fischeri is from the Vibrionacea family of marine proteobacteria which includes many species that have both beneficial and harmful relationship with animals (Ruby et al., 2005). It is a gram negative bioluminescent marine bacterium that forms mutually symbiotic

relationships with many species of fish and squids (Ruby et al., 1999). Vibrio fisheri is non-pathogenic for humans and grows best at 23C in the presence of three percent (3%) salt. Unlike other delicate organisms, it does not require an incubator or extensive media preparation (Thomulka & Peck, 1995). Vibrio fischeri is a motile bacterium and can be found free living on ocean or inside the microhabitats of its host light organs. Outside from its primary hosts, Vibrio fischeri can be found living freely as marine snow, in fecal pellets, as saprohytes (Herring and Widder, 2001), and amongst the microbial flora in the guts of marine animals. The heterotrophic bacteria are distributed in the pelagic zone of temperate and subtropical waters (Ruby

8
et al., 1999). The proteins required for the production of bioluminescence are determined in a set of genes called the lux operon. The bioluminescent bacteria produces light in a chemical reaction where luciferin, a substrate molecule, is oxidized by an enzyme, luciferase. It naturally occurs in the digestive tract and on the skin surface of marine animals. It is of symbiont with sepiolid squids (Cephalopoda: Sepiolidae) and monocentrid fishes (Actinopterygii: Monocentridae). Vibrio fischeri host specialists exhibit dominance within the allopatrically distributed Euprymna squid. In contrast, Sepiola squid contains sympatric species which gave rise to Vibrio fischeri host generalist (Soto et al., 2008). Continuous cultivation of Vibrio fischeri is similar to their natural growth mode in symbiotic relationships with fish or squid. In those relationships a large proportion of the bacterial population is regularly expelled from the light organ, while nutrients and oxygen are continuously provided by the host to support continuous replication of the bacteria (Scheerer et al., 2006). Temperature and salinity are integral agents in governing Vibrio population dynamics, physiological stress, and evolution. It may also alter colonization efficiency prior to infection (Soto et al., 2008). Fish as Highly Perishable Food Fish is the most perishable (Hammond, 2002) and consumed food (Ascalon et al., 2010). Fish spoilage results from three basic mechanisms: Enzymatic autolysis, oxidation, microbial growth (Ghaly et al., 2010). A

9
number of proteolytic enzymes are found in muscle and viscera of the fish after catch. These enzymes contribute to degradation

in fish muscle and fish products during storage and processing. There is a sensorial or product associated alteration that can be contributed by proteolytic enzymes (Engvang & Nielsen, 2001). During improper storage of whole fish, proteolysis is responsible for degradation of proteins and is followed by a process of solubilization (Lin & Park, 1996). On the other hand, peptides and free amino acids can be produced as a result of autolysis of fish muscle proteins, which lead towards

the spoilage of fish meat as an outcome of microbial growth and production of biogenic amines (Fraser & Sumar, 1998). Freshness Indicator. Its freshness can be detected using a sensor which is a device to differentiate the physical state and chemical condition of an object (Ascalon et al., 2010). The following methods are currently used for the detection of freshness: polyaniline, trimethylamine, and ammonia. Polyalinine is used as an optic sensor which can detect total volatile basic nitrogen which evolves when a fish starts to decay because of bacteria or enzymatic degradation (Ascalon et al., 2010). Trimethylamine is responsible for the bad odor of fish. Its presence is due to the bacterial diminution of trimethyl oxide which is naturally present in many marine organisms (Ascalon et al., 2010). A strong ammonia smell is the result of protein breakdown and indicates old product, extended freezer storage,

10
and possibly mishandled product by storage at elevated temperature above 4C (Hammond, 2002). Meat Quality Meat quality traits are closely related to the biological traits of the animal; like tenderness, water holding capacity of meat, muscle growth, development, and carcass composition (Bendixen, 2005). The quality control of meat is totally different from fish because it needs only a short period of time for maintaining its fresh state which is important for high quality. Meat quality is judged from eating quality which is best possible just before bacterial spoilage begins. The word meat freshness control should be regarded as a complex concept involving aging bacterial deterioration of meat (Funazaki et al., 1995). The development of protein oxidation in meat systems has been related to color and texture deterioration, DNPH (2, 4-

dinitrophenylhydrazine) method is used for evaluating protein oxidation (Nychas & Tassou, 1997). The lack of characterization of protein oxidation in foods might be due to the complexity of the protein target, the number of possible reactions on the protein backbone and side chain, the large diversity of modifications and the complexity of the mechanisms. However, there is now significant evidence that protein oxidation can also have a deleterious impact on fish quality (Ghaly et al., 2010).

11
The amount of glucose and lactose decreases with an increase of pseudomonads. The changes in the concentration of the free amino acids in meat samples stored under vacuum pack or aerobic conditions were used to monitor the microbial spoilage. Free amino acids increased in proportion to the colony counts (Nychas & Tassou, 1997). Accumulation of lactic acids will lead to temperature increase, which, coupled with pH loss that would result to protein denaturation. This denaturation will then be the cause of loss of protein solubility, water holding capacity and the muscles coloration (Maltin et al., 2003). During post mortem, lactic acid is produced due to anaerobic metabolism brought about by the depletion of oxygen, pH falls, and eventually, all defense mechanism would fail. If pH continues to drop, from 7 to 5.6-5.7 in 6-8 hrs post mortem and pH of 5.3-5.7 in 24 hrs post mortem (Flores et al., 2000), it will activate cathepsins and lead to proteolysis (Kwasiborksi et al., 2008). Proteases and Proteolysis Organisms growing on the muscle of meat secrete extracellular enzyme protease which can degrade substrate causing damage to the muscles. It is well known that synthesis of microbial proteases is influenced by the availability of nutrients while the presence of protein is not required for protease synthesis. Indeed, it was proposed that (i) organisms generate very low basal levels of extracellular enzymes in the absence of an inducer, and (ii) the regulation and extracellular production of proteinases are based

12
on induction and catabolite repression (Nychas & Tassou, 1997). Proteolysis is the degradation of proteins by proteases, and is due to pseudomonads. It occurs when the bacterial population reached log 10 7-8 cfug-1 and during the depletion of glucose concentration. As long as low molecular-weight components are available especially glucose, meat proteolysis is inhibited. The levels of protein or fat do not change during the onset of rigor nor are they substrates for microbial attack prior to the onset of spoilage (Nychas & Tassou, 1997). Biogenic Amines Biogenic amines are low molecular weight organic bases that are synthesized and degraded during the cellular metabolism activities inmicroorganisms, plants and animals. It is found in foods with high levels of protein like meat (Onal, 2007). Biogenic amine determination in meat is appropriate for detecting early spoilage and their quantities can be related to the freshness of the meat (Vinci & Antonelli, 2002). Biogenic amines in foods and beverages are formed by microbial decarboxylation of the corresponding amino acids. It was also found that biogenic amines are not significantly reduced by high temperature treatment (Saaid et al., 2009). The variation of the biogenic amine differed quite markedly between white and red meats. Cadaverine was the amine produced in the maximum quantity in both meats. This amine appeared sooner and increased more quickly in white meat. Serotonin was present in red meat in a very low

13
quantity and was completely absent in white meat. In white meat, all the biogenic amine increased faster than red meat, probably because of shorter muscle fibres, which can be easily attacked by proteolytic enzymes (Vinci & Antonelli, 2002). Biogenic amine in food can cause headaches, nausea and palpitations. Excess in tyramine could cause hypertension, meanwhile serotonin is a vasoconstrictor (Vinci & Antonelli, 2002).

14

Chapter III METHODOLOGY

Preparation of Experimental Organism For the preparation of luminous bacteria agar, 9 grams of NaCl, 1.5 grams of yeast extract powder, 4.5 grams of tryptone, and 5.4 grams of Bacto agar were prepared and were mixed with 300 milliliters of distilled water. The prepared agar was autoclaved at 121C for 15 minutes. It was then transferred to the corresponding sterile plates where it was plated to be used for culturing. The prepared sterile agar plates were streaked with the bacteria, Vibrio fischeri taken from University of Santo Tomas Collection of Microbial Strains, at room temperature. The plates were then incubated at room temperature incubation period of 24 hours. After 24 hours, the bacteria were then subcultured to prolong their generations.

Preparation of Standardized Vibrio fischeri Saline Solution A heavy and brightly luminous suspension of Vibrio fischeri was prepared from a 20-hour old plate culture. A 3% saline was poured over the agar plate and the colonies were scraped off gently using a sterile wire loop. The turbid suspension was poured in a 500 mL flask, was swirled to

15
effect homogenization, and was subsequently adjusted to McFarland # 2 Barium Sulfate Turbidity Standard (0.2 mL 1% w/v BaCl2 and 9.8 mL 1% v/v H2SO4). Preparation of the Pork / Fillet Sample One kilo of ground pork and one kilo of fish fillet from Decapterus macrosoma (Galunggong) were bought from SM Supermarket, Fairview, Quezon City. They were then laid exposed on a flat wrapping plastic to a room temperature for 24 hours which was noted as time zero of the incubation period. Portions of the pork/fillet at 100 grams weight were aseptically removed from the meat as the need arose using flame sterilized tweezers and scalpel. The 100 gram pork/fillet were collected from the meat, placed on aluminum foils, and stored in the freezer to arrest growth of resident microbes at time intervals of 3 hours totaling 8 pork/fillet samples for a period of 24 hours. Using this procedure, the first pork/fillet sample was incubated at room temperature for 3 hours and the last pork/fillet sample was no longer stored in the freezer and received incubation period of 24 hours at room temperature. Each pork/fillet sample was mixed with 10 mL sterile 3% w/v sodium chloride solution in distilled water contained in 250 mL Erlenmeyer flasks. Each pork/fillet samples pH was noted with the use of pH meter.

16
Tube Bioluminescence Extinction Technology (TuBET) Volumes of 1 mL of McFarland #2 standardized Vibrio fischeri bacterial saline suspension were added individually to all the 24 tubes containing 3 mL of the meat/fillet suspension. Each tube was immediately capped and the 2 tubes in a row were grasped together, inverted once and returned back in the rack. Inversion mixes the contents of the tubes causing them to shine homogeneous bright bluish-green in the dark. The time it took for each luminous tube to black out was noted down and recorded. A blackout is defined as the first sign of the disappearance of the homogeneity of bioluminescence contained in a tube. Blacking out usually start at the bottom and central portion of the tube spreading upward until only a thin luminous part remains at the top most portion. Breeds Method of Direct Cell Counting A 0.1 mL from 100g/10 mL pork/fillet saline suspension was spread uniformly using the inoculating loop covering 15 mm x 15 mm area. The smear was fixed by heating and stained with crystal violet. The smear was examined under Oil Immersion Objective. Cells in 10 microscopic fields were directly counted. Each field was counted per quadrant and was multiplied by four to represent the whole field. The counting of total number of cells was determined by calculating the total number of microscopic

17
fields per 15 mm x 15mm of the smear. The total number of cells can be counted with the equation:

Data Processing and Statistical Analysis The collected data: direct cell count, pH, and time of TuBET were analyzed using correlation coefficient and method of least squares or regression equation.

18

Flow Chart of Sample Preparation for TuBET

Fish Fillet / Ground Pork Incubation of Samples every 3 hours for 24 hours

Test organism obtained from UST CMS

Fish Fillet/ Ground Pork Samples Mixed with Saline

Culturing of Vibrio fisheri in T-YE-S Agar Medium

Direct Cell Counting by Breeds Method of Ground Pork and Fish Fillet Samples

Preparation of 1 mL Vibrio fisheri saline suspension

pH Determination Using pH Meter

Transferring of 3 mL saline suspension of meat / fillet samples in test tubes

Determination of Time of Bioluminescence Extinction per tube

Data Processing and Statistical Analysis

19

Chapter IV RESULTS AND DISCUSSIONS

Figure 1 shows the inverse relationship of bacterial cell count against the tube bioluminescence extinction for ground pork. The graph showed that the period of bioluminescence extinction decreased as the bacterial count per gram of ground pork increased. This is due to the high demand for oxygen by the rapidly growing bacteria in the pork samples. Consequently, this also leads to the rapid formation of biogenic amines (Vinci & Antonelli, 2002) which exert some degree of toxicity to the luminous bacteria causing the bioluminescence to undergo extinction. The graph shows a high degree of linearity with a correlation coefficient of 0.9433, which can then be used to predict the number of bacterial cells present in the pork sample. Since the maximum allowable number of bacteria (cells/g) is 10 million which is recommended by the International Commission on the Microbiological Specifications of Foods (ICMSF) (Fiorentini et al., 2001), the time of bioluminescence extinction is therefore 104 seconds. Thus, a pork sample that undergoes 104 seconds extinction is not anymore suitable for human consumption. Beyond the 9 th hour, the ground pork was already considered spoiled or lacks freshness.

20
Moreover, the shortest possible time of light extinction based on the regression equation of the graph, y = -3.903x + 43.2, is 104 seconds.
y = -3.903x + 143.28 R = 0.9433 140 Tube Bioluminescence Extinction (seconds)
(3 hrs)

120
(6 hrs)

100 80

(12 hrs) (9 hrs) (18 hrs) (15 hrs)

60 40 20 0 0 10 20 30 40 Bacterial Cell Count (cells/g x 1 million)

(21 hrs) (24 hrs)

Fig. 1. Tube Bioluminescence Extinction Time vs. Bacterial Cell Count of Ground Pork

Figure 2 shows the inverse relationship of

time of tube

bioluminescence extinction against bacterial cell count of fish fillet from Decapterus macrosoma. At the 9th hour, the bacterial cell count of fish fillet was 1.24 x 107 cells/g, which indicates that it already undergone spoilage based on the recommendation of ICMSF which is 1 x 107 cfu/g. With the regression equation of y = -0.9201x + 391.0, the shortest possible time of bioluminescence extinction is 299 seconds with a bacterial

21
cell count of 1 x 107. The two variables were highly correlated in an inversely linear fashion due to a correlation coefficient value of 0.967. Although the ground pork and fish fillet have approximately similar count of bacterial cells per gram, 7.06 X 106 and 7.42 X 106 respectively, the time of tube bioluminescence extinction of fish fillet which is 317 seconds, is longer in contrast to the ground pork, which has a bioluminescence time extinction of 113 seconds. The longer period top achieve bioluminescence extinction in fish compared to pork can be attributed to the saltwater/marine habitat of Galunggong which is favorable to the marine nature of Vibrio fischeri (Ruby et al., 1999).

y = -0.9201x + 391.0 R = 0.967 Tube Bioluminescence Extinction (seconds) 350 300 250 200 150
(21 hrs) (12 hrs) (3 hrs) (6 hrs) (9 hrs) (15 hrs) (18 hrs)

100
(24 hrs)

50 0 0 5 10 15 20 25 30 35 40 Bacterial Cell Count (cells/g x 1 million)

Fig. 2. Tube Bioluminescence Extinction vs. Bacterial Cell Count of Fish Fillet from Decapterus macrosoma

22
Figure 3 shows the time of exposure of ground pork at room temperature and the corresponding increase in pH. During the first three hours, the pork samples pH was acidic at 5.3 since during the early stages after slaughter; lactic acid accumulates in the muscles due to fermentation, an anaerobic metabolism brought about by the depletion of oxygen (Flores, et al., 2000). The subsequent increase in the pH of pork samples is due to the action of bacteria and autolytic enzymes present in the pork. Bacteria are responsible for the spoilage of proteins in the pork as they breakdown the proteins into soluble peptides and peptides into amino acids. The amino acids are then decarboxylated by enzymes to produce numerous volatile amines which are known for their obnoxious odor of decay and strongly basic chemical property in aqueous medium thereby rendering the medium alkaline. Spoilage of protein rich foods like pork and fish is commonly determined organoleptically by their texture, aroma, and color. As the pH continues to become basic, the carboxylic functional group of amino acids is abstracted by decarboxylases produced by the rapidly growing bacteria and in the process generate large amount of biogenic amines. These amines are toxic to the bioluminescent bacteria causing them to lose bioluminescence when exposed to; therefore, an increasing amount of biogenic amine would mean shorter period of bioluminescence extinction.

23
A shorter period to achieve bioluminescence extinction means higher bacterial density in the fresh food sample and higher bacterial density means less freshness and a higher degree of spoilage.
30 25 20 15 10 5 0 4 5 6 7 8 pH 9 10 11 12

Fig. 3. Time of Exposure to Room Temperature vs. pH of Ground Pork

Figure 4 shows the pH of fish fillet having acidic properties during the first six hours while gradually exhibiting basicity on the next few hours. The graph exhibited a direct relationship between the time of exposure to room temperature and the pH of fish fillet. At high concentration of bacteria, there was a high pH that caused the bioluminescent bacteria to shorten their time period. Numerous biogenic amines including tryptamine, putrescine, cadaverine, serotonin,

Time of exposure to room temperature (hours)

24
tyramine, spermidine,and spermine etc. will be produced resulting in the inhibition of the bioluminescence.

30 Time of exposure to room temperature (hours) 25 20 15 10 5 0 5 6 7 8 pH 9 10 11 12

Fig. 4. Time of Exposure to Room Temperature vs. pH of Fish Fillet from Decapterus macrosoma

Since a bacterium is a unicellular organism; therefore, a colony of bacteria is considered to a cell. Thus, cells/g and cfu/g were used similarly in the discussion. Table 1 shows the different methods used by the markets in determining freshness of meat products from FAO (Civera et al.,1995). DEFT or Direct Epifluorescence technique requires a fluorescence microscope and ATP requires a luminometer, which are expensive pieces of instrument, while TuBET does not rely on any sophisticated laboratory

25
instrument since the blacking out of tubes can readily be observed in a dark room. Moreover, Plate count and Petrifilm have higher sensitivities than TuBET but require much longer incubation period of 3 to 5 days and are also quite expensive. TuBETs sensitivity of 106-107 cells/g is lower in comparison with plate count and petrifilm methods, which can detect bacteria at low count of 10 cfu/g. However, the bacterial cell count/g of TuBET of 106 107 is within the range limit set by the ICMSF for acceptable bacterial count in foods. A bioluminescence extinction period of 3 minutes and 5 minutes for ground pork and fish fillet respectively, is the limit of Freshness for these perishable foods when tested using TuBET. This time of bioluminescence extinction corresponds to a bacterial count/g or cfu/g of 10,000,000 the maximum allowable limit set by the ICMSF. Thus, TuBET can be used as an inexpensive, low cost method of determining bacterial density and freshness of perishable foods like pork and fish.

26
Table 1. Comparison of TuBET to Different Methods in Determining Freshness of Meat Products

Methods of the Determination for Freshness of Meat Products

Plate Count or Iron agar Redigel/Petrifilm
TM

Temperature (C)
15 25 15 25 15 30

Incubation Period
3 5 days 3 5 days 3 4 hours

Sensitivity

References

10 (cfu/g) 10 (cfu/g) 10 10 (cfu/g)

4 5

FAO FAO FAO

SM

Microcolony DEFT

DEFT

30 min.

10 10 (cfu/g)

FAO

ATP

1 hour

10 10 (cfu/g)

FAO

Limulus Lysate Test

2 3 hours

10 10 (cfu/g)

FAO

Microcalorimetry / Dye Reduction Conductance/Capacitance Tube Bioluminescence Extinction Technology (TuBET) for ground pork Tube Bioluminescence Extinction Technology (TuBET) for Decapterus macrosoma

15 25

4 40 hours

10 (cfu/g)

FAO

Room temperature

1-3 min.

10 - 10 cells/g

This Study

Room temperature

1-5 min.

10 - 10 cells/g

This Study

27

Chapter V CONCLUSIONS AND RECOMMENDATIONS

Conclusions There is a gradual increase in bacterial cell count of room temperature incubated fresh ground pork and fish fillet over a period of time. This is defined as an inverse linear relationship between the cells/g and bioluminescence extinction time of both ground pork and fish fillet with correlation coefficients of 0.9433 and 0.9675 respectively. There was also an observed increased in the pH of the meat samples over time at room temperature. This observation can be attributed to the production of biogenic amines, alkaline, and volatile compounds. TuBET exhibited the shortest time of assay in comparison to other methods/techniques in the determination of the freshness of foods but has a low sensitivity of 106 - 107 cells/g. With its shorter time of bioassay, simplicity, and cost effectiveness, Tube Bioluminescence Extinction Technology (TuBET) was indeed proven as an excellent method for the rapid determination of the freshness of highly perishable foods like pork and fish.

28
Recommendations Firstly, it is recommend that TuBET be used to determine the freshness of other perishable foods like ground beef, chicken and other sea foods like prawns and squids. Secondly, to determine the Double Dead status of pork and beef using TuBET. Thirdly, to determine the Freshness of other protein-rich processed foods like soya-based Tofu and related products. Lastly, to test the efficacy of TuBET for use in the local wet markets for fast and inexpensive determination of freshness of meat products.

29

Chapter VI REFERENCES Ascalon, K., Gomez, K. J., Santos, O.M., Quinto, E. (2010). Evaluation of Polyalinine Sensor for Food Freshness of Galunggong (Decapterus macarellus). Sensor & Analytical Devices (Session1-8), 2010 UST Undergraduate Symposium, Research Center for Natural Sciences. 32. Bendixen, E. (2005). The Use of Proteomics in Meat Science. Meat Science 71, 138149. Flores, M., Moya, V. J., Aristoy, M. C., & Toldra, F. (2000). Nitrogen Compounds as Potential Biochemical Markers of Pork Meat Quality. Food Chemistry 69, 371-377. Funazaki, N. et al., (1995). Application of Semiconductor Gas Sensor to Quality Control of Meat Freshness in Food Industry. Sensors and Actuators B 24-25, 797-800. Ghaly, A.E., D. Dave, S. Budge and M.S. Brooks, (2010). Fish Spoilage Mechanisms and Preservation Techniques: Review. Am. J. Applied Sci., 7: 859-877.

30
Hammond, J., Marquis, B., Michaels, R., Oickle, B., Segee, B., Vetelino, J., Bushway, A., Camire, M. E., & Davis-Dentici, K. (2002). A SemiConducting Metal-Oxide Array for Monitoring Fish Freshness. Sensors and Acuators 84, 113-122. Hernando, M., Malato, O., Farr, M., Fernandez-Alba, A., & Barcel, D. (2006). Application of Ring Study: Water Toxicity Determinations by Bioluminescence Assay with Vibrio fischeri. Talanta, 370-376. Hutchens, M., & Luker, G. D. (2007). Applications of Bioluminescence Imaging to the Study of Infectious Diseases. Cellular Microbiology, 23152322. Kwasiborski, A., Sayd, T., Chambon, C., Sant-Lhoutellier, V., Rocha, D., & Terlouw, C. (2008). Pig Longissimus Lumborum Proteome: Part II: Relationships Between Protein Content and Meat Quality. Meat Science 80, 982996. Lappalainen, J., Juvonen, R., Nurmi, J., & Karp, M. (2001). Automated Color Correction Method for Vibrio fischeri Toxicity Test.

Comparison of Standard and Kinetic Assays. Chemosphere, 635641. Maltin, C., Balcerzak, D., Tilley, R., & Delday, M. (2003). Determinants of Meat Quality: Tenderness. Proceedings of the Nutrition Society 62, 337-347.

31
Nealson, K.H, & Hastings, J.W. (1992). The Luminous Bacteria. In: A. H. Balows, G. Trueper, M. Dworkin, W. Harder and K.H. Schleifer (eds.) The Prokaryotes. A Handbook on the Biology of Bacteria, Ecophysiology, Isolation, Identification and Applications.2 nd ed. Springer-Verlag. Nychas, G. E. & Tassou, C. (1997). Spoilage Processes and Proteolysis in Chicken as Detected by HPLC. J Sci Food Agric 74, 199-208. Ocampo-Duque, W., Sierra, J., Ferr-Huguet, N., Schuhmacher, M., & Domingo, J. L. (2008). Estimating the Environmental Impact of Micro-pollutants in the Low Ebro River (Spain): An Approach Based on Screening Toxicity with Vibrio fischeri. Chemosphere, 715721. Onal, A. (2007). A Review: Current Analytical Methods for the Determination of Biogenic Amines in Foods. Food Chemistry 103, 14751486. Perez-Juan, M., Flores, M., & Toldra, F. (2008). Effect of Pork Meat Proteins on the Binding of Volatile Compounds. Food Chemistry 108, 12261233. Ruby, E. G., & McFall-Ngai, M. J. (1999). Oxygen-utilizing Reactions and Symbiotic Colonization of the Squid Light Organ by Vibrio fischeri. Trends in Microbiology 7, 414-420.

32
Saaid, M., Saad, B., Hashim, N. H., Mohamed Ali, A. S., & Saleh, M. I. (2009). Determination of Biogenic Amines in Selected Malaysian Food. Food Chemistry 113, 13561362. Scheerer, S., Gomez, F., & Lloyd, D. (2006). Journal of Microbiological Methods. Bioluminescence of Vibrio fischeri in Continuous Culture: Optimal Conditions for Stability and Intensity of Photoemission 67, 321-329. Soto, W., Gutierrez, J., Remmenga, M.D., & Nishiguchi, M.K. (2009). Salinity and Temperature Effects on Physiological Responses of Vibrio fischeri from Diverse Ecological Niches. Microb Ecol 57, 140150. Thomulka, K., & Peck, L. (1995).Use of Bioluminescence in Detecting Biohazardous Substances in Water, Philadelphia College of Pharmacy and Science 215, 596. Vinci, G., & Antonelli, M.L. (2002). Biogenic Amines: Quality Index of Freshness in Red and White Meat. Food Control 13, 519524.

33

APPENDIX I Bacterial Cell Count Raw Data of Ground Pork (One Quadrant)
Slide Number 1B 1C 2A 2B 2C 3A 3B 3C 4A 4B 4C 5A 5B 5C 6A 6B 6C 7A 7B 7C 8A 8B 8C Field 1 80 101 225 272 150 363 287 342 509 381 397 530 539 520 750 699 738 886 928 817 1041 1005 1128 Field 2 121 90 238 250 182 282 283 348 444 392 385 488 567 499 782 730 778 897 934 796 995 1266 1172 Field 3 95 79 199 212 290 299 277 351 476 365 443 625 554 472 682 767 708 932 899 863 1171 1234 1198 Field 4 77 88 187 243 239 344 362 282 390 459 472 388 433 468 762 665 648 879 967 971 1300 997 1246 Field 5 111 69 266 158 281 387 338 274 525 482 430 559 582 545 724 747 606 924 830 948 950 1088 1385 Field 6 155 74 305 192 279 365 348 387 488 464 451 601 538 581 658 677 668 967 864 893 977 1127 1348 Field 7 143 92 277 220 268 355 435 391 435 438 412 598 577 537 708 683 725 881 905 909 1045 1239 1422 Field 8 188 100 190 288 251 379 401 433 482 469 474 533 452 466 763 633 779 945 857 922 1242 1426 1378 Field 9 105 119 206 263 247 321 298 422 474 499 486 525 489 491 756 711 648 872 788 937 1189 987 1246 Field 10 125 166 233 242 207 309 253 306 545 372 495 548 522 530 722 694 785 881 821 855 1086 1184 993

Note: Slide number in accordance to the hours of exposure in room temperature: Slide number 1 = 3 hours Slide number 4= 12 hours Slide number 7= 21 hours Slide number 2 = 6 hours Slide number 5 = 15 hours Slide number 8 = 24 hours Slide number 3= 9 hours Slide number 6= 18 hours

34

APPENDIX II Bacterial Cell Count Raw Data of Ground Pork (One Quadrant x 4)
Slide Number 1B 1C 2A 2B 2C 3A 3B 3C 4A 4B 4C 5A 5B 5C 6A 6B 6C 7A 7B 7C 8A 8B 8C Field 1 320 404 900 1088 600 1452 1148 1368 2036 1522 1588 2120 2156 2080 3000 2796 2952 3544 3712 3268 4164 4020 4512 Field 2 484 360 952 1000 728 1128 1132 1392 1776 1568 1540 1952 2268 1996 3128 2920 3112 3588 3736 3184 3980 5064 4688 Field 3 380 316 796 848 1160 1196 1108 1404 1904 1460 1772 2500 2216 1888 2728 3068 2832 3728 3596 3452 4684 4936 4792 Field 4 308 352 748 972 956 1376 1448 1128 1560 1836 1888 1552 1732 1872 3048 2660 2592 3516 3868 3884 5200 3988 4984 Field 5 444 276 1064 632 1124 1548 1352 1096 2100 1928 1720 2236 2328 2180 2896 2988 2424 3696 3320 3792 3800 4352 5540 Field 6 620 296 1220 768 1116 1460 1392 1548 1952 1856 1804 2404 2152 2324 2632 2708 2672 3868 3456 3572 3908 4508 5392 Field 7 572 368 1108 880 1072 1420 1740 1564 1740 1752 1648 2392 2308 2148 2832 2732 2900 3524 3620 3636 4180 4956 5688 Field 8 752 400 760 1152 1004 1516 1604 1732 1928 1876 1896 2132 1808 1864 3052 2532 3116 3780 3428 3688 4968 5704 5512 Field 9 420 476 824 1052 988 1284 1192 1688 1896 1996 1944 2100 1956 1964 3024 2844 2592 3488 3152 3748 4756 3948 4984 Field 10 500 664 932 968 828 1236 1012 1224 2180 1488 1980 2192 2088 2120 2888 2776 3140 3524 3284 3418 4344 4736 3972

Note: Slide number in accordance to the hours of exposure in room temperature: Slide number 1 = 3 hours Slide number 4 = 12 hours Slide number 7 = 21 hours Slide number 2 = 6 hours Slide number 5 = 15 hours Slide number 8 = 24 hours Slide number 3 = 9 hours Slide number 6 = 18 hours

35

APPENDIX III Descriptive Analysis of Ground Pork Using the Breeds Method
Slide Number Hours Exposed to Room Temperature 3 6 9 12 15 18 21 24 Time (seconds) pH Average Cell Count per mL 6 (x10 ) 32.7 70.6 102.2 135 157.6 214 267.7 350.6 cells/g 6 (x10 ) SD on Average

1 2 3 4 5 6 7 8

131 113 99 98 78 77 21 10

5.3 6.0 6.9 7.8 8.5 9.1 9.9 10.5

435.6 436 941.33 941 1362.93 1363 1804.47 1804 2100.93 2101 2852.8 2853 3569 4675.33 4675

3.27 7.06 10.2 13.5 15.7 21.4 26.8 35.1

62.79 14.36 50.81 92.39 57.20 62.55 54.36 307.59

36

APPENDIX IV

Bacterial Cell Count Raw Data of Fish Fillet from Decapterus macrosoma (One Quadrant)
Slide Number 1A 1B 1C 2A 2B 2C 3A 3B 3C 4A 4B 4C 5A 5B 5C 6A 6B 6C 7A 7B 7C 8A 8B 8C Field 1 178 204 198 298 326 331 398 406 426 512 522 536 599 618 626 712 723 731 797 826 862 997 1167 1249 Field 2 194 226 218 287 334 299 412 424 433 532 527 510 622 631 632 722 719 728 813 831 873 1135 1236 1138 Field 3 234 198 226 314 298 324 426 399 412 497 528 512 618 629 622 731 727 738 826 838 851 998 1134 1342 Field 4 197 238 214 332 321 297 395 415 429 508 526 514 632 621 618 718 732 729 824 842 843 1305 1187 1256 Field 5 235 214 226 292 329 340 417 395 420 522 499 534 629 638 615 729 740 736 819 836 878 1286 1231 1321 Field 6 225 206 213 314 289 310 425 419 432 489 518 522 636 639 623 731 739 746 832 844 851 1176 1324 1189 Field 7 186 229 199 328 319 322 389 422 439 513 526 528 618 597 634 724 736 732 856 863 882 1154 1197 1276 Field 8 217 196 227 340 328 318 381 413 399 521 507 531 602 619 631 740 748 737 829 831 876 1233 1267 1305 Field 9 197 203 219 331 298 315 392 429 415 499 512 522 607 623 640 731 747 742 849 835 841 1174 1208 1298 Field 10 228 209 232 297 314 328 413 432 404 508 526 517 628 636 621 729 741 746 834 828 856 1204 1186 1301

Note: Slide number in accordance to the hours of exposure in room temperature: Slide number 1 = 3 hours Slide number 4 = 12 hours Slide number 7 = 21 hours Slide number 2 = 6 hours Slide number 5 = 15 hours Slide number 8 = 24 hour Slide number 3 = 9 hours Slide number 6 = 18 hours

37

APPENDIX V

Bacterial Cell Count Raw Data of Fish Fillet from Decapterus macrosoma (One Quadrant x 4)
Slide Number 1A 1B 1C 2A 2B 2C 3A 3B 3C 4A 4B 4C 5A 5B 5C 6A 6B 6C 7A 7B 7C 8A 8B 8C Field 1 712 816 792 1192 1304 1324 1592 1624 1704 2048 2088 2144 2396 2472 2504 2848 2892 2924 3188 3304 3448 3988 4668 4996 Field 2 776 904 872 1148 1336 1196 1648 1696 1732 2128 2108 2040 2488 2524 2528 2888 2876 2912 3252 3324 3492 4540 4944 4552 Field 3 936 792 904 1256 1192 1296 1704 1596 1648 1988 2112 2048 2472 2516 2488 2924 2908 2952 3304 3352 3404 3992 4536 5368 Field 4 788 952 856 1328 1284 1188 1580 1660 1716 2032 2104 2056 2528 2484 2472 2872 2928 2916 3296 3368 3372 5220 4748 5024 Field 5 940 856 904 1168 1316 1360 1668 1580 1680 2088 1996 2136 2516 2552 2460 2916 2960 2944 3276 3344 3512 5144 4924 5284 Field 6 900 824 852 1256 1156 1240 1700 1676 1728 1956 2072 2088 2544 2556 2492 2924 2956 2984 3328 3376 3404 4704 5296 4756 Field 7 744 916 796 1312 1276 1288 1556 1688 1756 2052 2104 2112 2472 2388 2536 2896 2944 2928 3424 3452 3528 4616 4788 5104 Field 8 868 784 908 1360 1312 1272 1524 1652 1596 2084 2028 2124 2408 2476 2524 2960 2992 2948 3316 3324 3504 4932 5068 5220 Field 9 788 812 876 1324 1192 1260 1568 1716 1660 1996 2048 2088 2428 2492 2560 2924 2988 2968 3396 3340 3364 4696 4832 5192 Field 10 912 836 928 1188 1256 1312 1652 1728 1616 2032 2104 2068 2512 2544 2484 2916 2964 2984 3336 3312 3424 4816 4744 5204

Note: Slide number in accordance to the hours of exposure in room temperature: Slide number 1 = 3 hours Slide number 4 = 12 hours Slide number 7 = 21 hours Slide number 2 = 6 hours Slide number 5 = 15 hours Slide number 8 = 24 hours Slide number 3 = 9 hours Slide number 6 = 18 hours

38

APPENDIX VI

Descriptive Analysis of Fish Fillet from Decapterus macrosoma Using the Breeds Method

Slide Number

Hours Exposed to Room Temperature 3 6 9 12 15 18 21 24

Time (seconds)

pH

Average

Count per mL 6 (x10 ) 74.2 94.7 124.1 154.6 187.2 219.8 252.6 364.7

cells/g 6 (x10 )

SD on Average

1 2 3 4 5 6 7 8

317 309 298 224 216 208 142 60

6.1 6.7 7.4 8.2 9.2 9.8 10.3 10.8

990.40 1263.07 1654.80 2062.40 2496.53 2931.20 3368.80 4863.20

7.42 9.47 12.41 15.46 18.72 21.98 25.26 36.47

16.32 10.22 32.73 19.29 115.25 21.29 68.84 202.73

39

APPENDIX VII

Meat Saline Suspension Set-up

TuBET Set-up

Standardized Vibrio fischeri Saline Suspension

Subcultured Vibrio fischeri on T-YES Agar Medium

40

Microbes on a 6-hour Ground Pork Sample Observed on LPO lens, Stained with Crystal Violet

Microbes on a 3-hour Ground Pork Sample Observed on OIO lens, Stained with Crystal Violet