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217932034364SCHOOL OF FISHERIES AND AQUACULTURE SCIENCE
UNIVERSITI MALAYSIA TERENGGANU
BACHELOR OF APPLIED SCIENCE (FISHERIES)
0SCHOOL OF FISHERIES AND AQUACULTURE SCIENCE
UNIVERSITI MALAYSIA TERENGGANU
BACHELOR OF APPLIED SCIENCE (FISHERIES)

PROPOSAL
FINAL YEAR PROJECT
IDENTIFICATION OF LACTIC ACID BACTERIA BY USING STINGLESS BEE HONEY AND THE ANTIBACTERIAL EFFECT AGAINST SPOILAGE BACTERIA
LECTURER :DR. NURUL ULFAH BINTI ABDUL KARIM
PREPARED BY :NURHIDAYAH AZMIRAH BINTI MOHD JAMIL
(S43492)
INTRODUCTION
Background study
Fish is an important source of protein for human consumption. According to the Food and Agriculture Organisation (FAO) of the United Nations (1997), fish composed 16% of animal protein consumed by the world’s population. However, due to infection of pathogenic bacteria and intoxication, consumption of ?sh such as may cause diseases and food poisoning to the consumer. Some of these diseases that present on the external surfaces of fish including slime, gills and gut are caused by microorganisms (M. Karthiga Rani, et al., 2014). One of the fish species that are affected by the pathogenic bacteria is Oreochromis niloticus, which is one of the most important commercial fish in the world. Pathogenic and potentially pathogenic bacteria associated with fish include Clostridium botulinum, Listeria monocytogenes, Staphylococcus aureus, Salmonella species, Shigalla species, Escherichia coli and others (Pech et al., 2017). All of the harmful microorganism could enter the seafood processing chain due to poor standards of hygiene and sanitation during incorrect handling or storage. There is an urgent need to develop microbial control strategies, since diseases epidemics are recognized as limitation to fish production. Thus, Lactic Acid Bacteria (LAB) by using stingless bee honey can be used to face many parasites and pathogens to prevent disease in this study. LAB are important for preservation of nutritional qualities of the raw materials by extending shelf life, enhance the flavour and inhibit the spoilage and pathogenic bacteria on fish production (Nor Hazwani Mohd Hasali, et al., 2015).

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Problem statements
This study are seeks to identify Lactic Acid Bacteria (LAB) in stingless bee honey, identify the spoilage bacteria on Oreochromis niloticus and to determine the antibacterial effect on spoilage bacteria by using LAB in stingless bee honey. Fish composed of one of the sources of proteins, vitamins, minerals and rich in nutrients required for human growth. However, intensive growth of industry may cause contamination of fish and may affect not only the health of fish, but also raise safety concerns regarding to the fish as human consumption. It is well known that fish and fish products are often associated with human diseases. Thus, it is necessary to study the widespread presence of pathogenic bacteria and inhibition of microbial infection to ensure the safety of raw fish products. One of the effective initiative is by using LAB strain in stingless bee honey as honey is one of the oldest natural medicines known which used to treat illness, injuries and diseases.
Significant of studies
The most importantly, this study helps to ensure the proactive approach that can be done to prevent, cure and inhibit the bacterial growth on fish to ensure that fish and fishery products are good and safe for human consumption. This is because fish plays an important role in human diets and it has higher commercial value globally. Human that feed on the contaminated fish that containing toxin and spoilage bacteria will facing body health problems and it possibly causing food poisoning and other illness that may contribute to death. Other than that, this study is important to prevent the possible economic losses a fish farmer may suffer due to the disease occurrence on fish such as mortality, declining of fish growth and increase the time required for the fish to reach market weight-size. Next, the reduction and loss of Tilapia species, Oreochromis niloticus that cause by bacteria and parasitic diseases can be controlled. This is because Nile Tilapia is one of the most favoured culture fish species in the world because it is easy to be culture and have fast growing features. Moreover, by conducting this study, we can make sure the proactive approach by using Lactic Acid Bacteria (LAB) in stingless bee honey is helpful in the management of bacterial disease in fish.
Objectives
i. To isolate and identify Lactic Acid Bacteria on Stingless Honey bee
ii. To isolate and identify Spoilage bacteria on Oreochromis niloticusiii. To determine the antibacterial effect on spoilage bacteria
1.5 Justification
The research on antibacterial effect of stingless be honey (Kelulut honey) has not been conducted systematically in Malaysia, so little information is available.
Literature review
2.1 Habitat and biology
Oreochromis niloticus ( Linnaeus, 1758), which is known as Nile Tilapia is one of the world’s most important food fishes that come from family Cichlidae. This species can be found in freshwater and brackish habitat ( NBII & ISSG, 2008). It is widely introduced in aquaculture field because of it highly reproduction rates and easy to be culture. Based on the Global Invasive Species Database (ISSG), Oreochromis niloticus is the second most highly commercial farmed species in the world, which comes from one of the Tilapia family.
2.2 Taxonomy of Oreochromis niloticusKingdom Animalia
Phylum Chordata
Class Actinopterygii
Order PerciformesFamily CichlidaeGenus Oreochromis
Species Oreochromis niloticusFigure 1.0 Oreochromis niloticus 228600000
Oreochromis niloticus is a species of tropical fish belongs to the kingdom of Animalia, phylum Chordata and class Actinopterygii. This type of species is also known as Nile Tilapia (Snoeks, J., et al, 2018). O. niloticus are under the family of Cichlidae. Nile Tilapia is tolerant to high salinities and exist in a variety of freshwater and brackish habitats.

2.1.2 Morphology and reproduction

The Nile Tilapia, Oreochromis niloticus have a compressiform body shape with a cycloid scales. This species is omnivorous grazer, feeding mainly on phytoplankton, detritus and periphyton by ingesting the food sources through filter feeding. It has two types of teeth, which is pharyngeal teeth and jaws teeth. Both types were slightly different from one Tilapia species to another depending on their diet preferences. Nile Tilapia usually change the colour of it pectoral, caudal and dorsal fins into reddish during the spawning seasons (FAO, 2018).

The sexual reproduction of Nile Tilapia begins at 5 to 6 months. The male Tilapia will initiate the breeding by creating a spawning nest and the female will lays all of the eggs into the nest. Then, the female will carry the eggs in its mouth (mouth brooding) after all of the eggs had been fertilized by males. The female incubated and brooded it eggs in the mouth until hatching, approximately in 1 to 2 weeks (GISD,2018).

2.2 Stingless Bee Honey (Kelulut honey)
Honey is a natural sweetener that produced by honeybees mostly contain a mixture of sugar such as fructose and glucose, which provide it sweetness (Nor Hazwani et al., 2015). The physical properties and chemical composition of honey varies depending on the floral source from which the bees collect nectar (Pasupuleti et al., 2016). Stingless bees can be found in most tropical or subtropical region of the world. Stingless bee honey is a valuable bee product of the stingless bee. This precious bee product has traditionally been consumed directly and used in various nutritional and medical treatment since honey is known as the most effective natural product to treat a various kind of illnesses, diseases, and injuries. As the time passes, the belief that honey is rich in a nutrient has been carried into our days. Thus, an alternative medicine branch, called apitherapy, has been developed in recent years, where it’s offering treatment based on honey and other bee products against many diseases including bacterial infections. At present, a number of honeys are sold with standardized levels of other antibacterial medicinal product.  Stingless bees had become a popular phenomenon in Peninsular Malaysia in 2012 and there is proof that stingless bee honey contains medicinal properties and the bees can be cultivated as pollinators in the agricultural sector (Mohd Fahimee Jaapar et al., 2016). Stingless bee honey offers antibacterial activity due to the enzymatic production of hydrogen peroxide. It also helps to repair the damage tissue, which act as wound healing and its viscosity helps to provide a protective barrier to inhibit infection and diseases. In addition, honey is hygroscopic, which means that it can draw moisture out of the environment and dehydrate spoilage bacteria, since its contain high sugar value and low level of pH. According to Bulgasem Y. et al., 2016, honey is acidic with a pH between 3.2 and 4.5. Thus, this characteristic helps to prevent the growth of various pathogenic bacteria and fungi.

2.3 Lactic Acid Bacteria (LAB)
Lactic acid bacteria (LAB) which can be found in honey was characterized as Gram-positive bacteria that produced lactic acid in food fermentation and preservation. The presence of LAB in honey has been reported by the researcher when several strains from LAB such as L. Acidophilus had been isolated from honey and shows the antibacterial activities against Gram positive and Gram-negative bacteria (Aween et al., 2012).
2.4 Spoilage Bacteria
2.3 Analytic findings
3.0 Materials and Methods
3.0 Materials and Methods
3.1 Study area

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