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1 EVALUATION OF THE ANTIMICROBIAL AND SENSORY EFFECTS OF DRIED VINEGAR AND G UTTING ON SHELF LIFE EXTENSION OF FRESH MALAWIAN TILAPIA ( Oreochromis specie s) UNDER ICE STORAGE IN COOLERS By MATRINA MPEKETULA A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 201 3
2 201 3 Matrina Mpeketula
3 To my God and family
4 ACKNOWLEDGMENTS I would like to convey my gratitude to Dr. Sally K Williams my major professor for the extraordina ry guidance, encouragement and patience during the period of my studies and particularly in this research. I would also like to thank my committee members Dr. Adegbola T. Adesoga n and Dr. Geoffrey E. Dahl for their support, patience and advi c e during this project and all members of staff of the Department of Animal Sciences. I would also wish to convey my gratitude to Dr. Joe Yates and WTI Inc. for their support in providing the d ried white buffered v inegar used in the research. I would like to express thanks to my fellow graduate students for their suppor t. In a special way, I thank Ch e ryl Rock for being like my sister and a friend. I would like to express my love and appreciat ion to my husband Moffat Soko, and daughter Rumbie for the motivation, love and patience they offered me during my studies. Special acknowledgement should go to the USAID Initiative for Long Ter m Training and Capacity Building (UILTCB) program for the fin ancial support. Above all, I thank God
5 TA BLE OF CONTENTS ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 2 LITERATURE REVIEW ................................ ................................ .......................... 15 Geographical Location and Fishing Water Bodies in Malawi ................................ .. 15 The Fishing Industry i n Malawi ................................ ................................ ............... 15 Types of Fisheries in Malawi ................................ ................................ ............ 17 Small scale commercial sector ................................ ................................ .. 17 Large scale commercial sector ................................ ................................ .. 17 Fish Marketing in Malawi ................................ ................................ ........................ 18 Fish Trading ................................ ................................ ................................ ..... 18 Imported Fish and Fish Products ................................ ................................ ...... 19 Fish Processing in Malawi ................................ ................................ ...................... 19 Fresh Fish ................................ ................................ ................................ ........ 19 Further Processed Fish ................................ ................................ .................... 20 Sundried Fish ................................ ................................ ................................ ... 21 Smoked Fish ................................ ................................ ................................ .... 21 Parboiled Fish ................................ ................................ ................................ .. 22 Pan Roasting ................................ ................................ ................................ .... 22 Microbiology of Fresh Fish ................................ ................................ ...................... 22 Need for Shelf Stable Processed and Packaged Fish and Fish Products ............... 25 Fishery Products (non processed fish) ................................ ............................ 25 Current Preservation Processes in Malawi ................................ ............................. 26 Application of Antimicrobial Interventions for Control of Spoilage Bacteria in Tilapia ................................ ................................ ................................ .................. 27 3 MATERIALS AND METHODS ................................ ................................ ................ 30 Phase 1. Survey on Current Methods Employed by Fish Traders in Lilongwe, Limbe and Blantyre City Markets for Fresh Fish Quality Preservation in Coolers and the Effectiveness in Reducing Fresh Fish Spoilage ........................ 31 Phase 2. Evaluation of Dried Buffered Vinegar for Antimicrobial and Sensory Characteristics on Whole Un gutted and Gutted M alawian Tilapia Fish .............. 32 Fresh Fish Harvesting, Treatment and Evaluation ................................ ........... 32
6 Microbiological Analyses ................................ ................................ .................. 33 Sensory Analysis ................................ ................................ .............................. 34 Statistical Analysis ................................ ................................ ............................ 34 4 RESULTS AND DISCUSSION ................................ ................................ ............... 35 Phase 1. Results of Survey on Current Methods Employed by Fish Traders in Lilongwe, Limbe and Blantyre City Markets for Fresh Fish Quality Preservation in Coolers and the Effectiveness in Reducing Fresh Fish S poilage ................................ ................................ ................................ ............... 35 Survey Outcomes ................................ ................................ ............................. 35 Phase 2. Evaluation of Dried Buffered Vinegar for Antimicrobial Properties and Sensory Characteris tics on Whole Un gutted and Gutted Malawian Tilapia Fish ................................ ................................ ................................ ...................... 38 Microbiological Analyses ................................ ................................ .................. 38 Aerobic plate count ................................ ................................ .................... 38 Total coliform counts ................................ ................................ .................. 39 Generic E.coli counts ................................ ................................ ................. 39 Sensory Evaluation of Fresh Who le, gutted Oreochromis species ......................... 41 Sensory Panels for Cooked Fish ................................ ................................ ...... 41 Sensory Evaluation of Quality Attributes for Uncooked Oreoch romis species .. 41 Skin ................................ ................................ ................................ ............ 41 Odor ................................ ................................ ................................ ........... 42 Gills ................................ ................................ ................................ ............ 42 Flesh color ................................ ................................ ................................ 42 Eyes ................................ ................................ ................................ ........... 43 pH ................................ ................................ ................................ .............. 43 5 SUMMARY ................................ ................................ ................................ ............. 50 Conclusion ................................ ................................ ................................ .............. 52 APPENDIX A SURVEY QUESTIONNAIRE ................................ ................................ .................. 54 B SURVEY RESULTS SUMMARY ................................ ................................ ............ 59 C SCALE FOR EVALUATING QUALITY ATTRIBUTES OF FRESH MALAWIAN TILAPIA ( Oreochromis species ) STORED ON ICE IN COOLER BOXES FOR 5 DAYS ................................ ................................ ................................ ...................... 64 D FRESH FISH QUALITY ATTRIBUTE S SCORING SCALE FOR FRESH MALAWIAN TILAPIA ( Oreochromis species ) STORED ON ICE IN COOLER BOXES FOR 5 DAYS ................................ ................................ ............................. 65
7 E EXPERIMENTA L LAYOUT FOR DRIED BUFFERED VINEGAR ON APC OF FRESH MALAWIAN TILAPIA ( Oreochromis species ) STORED ON ICE IN COOLER BOXES FOR 5 DAYS ................................ ................................ ............. 66 LIST OF REFERENCES ................................ ................................ ............................... 67 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 72
8 LIST OF TABLES Table Page 4 1 Effects of dried buffered vinegar and gutting on aerobic plate counts of fresh Malawian Tilapia ( Oreochromis species ) stored on ice in cooler boxes for 5 days 44 4 2 Effect of gutting on APC of fresh Malawian Tilapia ( Oreochromis species ) treated with dried buffered vinegar and s tored on ice in cooler boxes for 5 days 45 4 3 Effect of DV on Total coliforms of gutted and ungutted fresh M alawian Tilapia ( Oreochromis species ) stored on ice in cooler boxes for 5 days 46 4 4 Effect of gutting and dried buffered vinegar treatment on E. coli of fresh Malawian Tilapia ( Or eochromis species) stored on ice in cooler boxes for 5 days 47 4 5 Effect of DV on pH of gutted and ungutted fresh Malawian Tilapia ( Oreochromis species ) stored on ice in cooler boxes for 5 days 48 4 6 Oreochromis species treated with dried buffered vinegar and stored on ice in coolers for 5 days 49
9 LIST OF ABBREVIATION S APC Aerobic Plate Count (or Aerobic Plate Counts) DV Dried White Buffered Vinegar EC Escherichia c oli (or Escherichia coli Counts) EU European Union FAO Fo od and Agriculture Organization GRAS Generally recognized as safe TCC Total Coliform Count (or Total Coliform Counts) TMA Trymethylamine TMAO Trymethylamine oxide US FDA United States Food and Drug Administration WHO World Health Organization K g Kilogram
10 Abstract of the Thesis Presented to the Graduate School Of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EVALUATION OF THE ANTIMICROBIAL AND SENSORY EFFECTS OF DRIED VINEGAR AND G UTTING ON SHELF LIFE EXTENSION OF FRESH MALAWIAN TILAPIA ( Oreochromis species ) UNDER ICE STORAGE IN COOLERS By Matrina Mpeketula May 2013 Chair : Sally K. Williams Major: Animal Sciences In Malawi, fish provides nutritional and economic benefits T hese benefits are reduced due to spoilage The objectives of this study were to identify knowledge gaps in fresh fish processing and handling among Malawian fish traders by administering a survey (Phase 1), and to determine antimicrobial and se nsory effects of dried buffered white vinegar ( DV ) and gutting on storage stability of fresh Malawian t ilapia (Phase 2) Whole f resh Tilapia was either gutted or left ungutted treated with 0 5, 8 or 1 0% (w/v) DV solutions and stored on ice in cooler boxes F ish were ana lyzed for pH, APC total coliform and g eneric E scherichia coli counts quality attributes on 0, 1 3 and 5 d storage. The DV treatment significantly reduced APC fecal coliforms and E.coli counts (P < 0.05) o n whole gutted and ungutted fresh fish up to 5 d storage and maintained fresh fish quality attribute s (P < 0.05) G utting of fish prior to DV treatment resulted in increased levels of APC This was especially true for 10% DV treatment. U ngutted fish treated with 5, 8 and 10% DV resulted in 2.65, 2.45 and 3.58 log reductions in APC,
11 respectively G utted fish treated with 5, 8 and 10% DV resulted in 2.25, 2.36 and 2.70 log reductions in APC, respectively. The pH of the fish ranged from 7.00 to 7.20 for all treatments and was not adversely affected (P > 0.05 ) by DV treatment s. This study revealed the need to provide access to food extension services, to enhance safety and quality
12 CHAPTER 1 INTRODUCTION In Malawi, fish plays a significant role both economically and nutritionally. However, due to high perish ability of fish and fish products, these economic and nutritional benefits are significantly reduc ed due to spoilage and reduced shelf life. In addition, the potential health risks of consuming contaminated fresh fish in conjunction with the high fish dema nd in areas far from local fishing grounds reveals the need for long term fish preservation methods in Malawi. T he fish consumption trend in Malawi includes consumption of various forms of fresh fish which has resulted in public health concerns that requ ir e very careful attention to ensure quality but also safety Because of minimal refrigeration and availability of ice, the onset of spoilage in fish in Malawi begins immediately after harves t and results in accelerated spoilage within 48 hours or less (As hie et al 1996). Spoilage refers to any change in the condition of food which makes it less acceptable, unacceptable or unsafe for human consumption. Spoilage may be accompanied by alterations in taste, smell, appearance or texture and is the result of c hemical and biological changes brought about in the fish postmortem due to enzyme and bacteria action (Ashie et al., 1996) The digestive enzymes cause extensive autolysis which results in meat softening, rupture of the belly wall and loss of blood, water and oil (Hall, 1997). Bacteria in the gut gills and skin increase and begin to metabolize surrounding low molecular weight compounds producing the volatile compounds associated with spoilage (Ashie et al., 1996). These compounds include Total Volati le Ba sic Nitrogen (TVB N) that consists of primary, secondary and tertia r y
13 amines TBA reactive substances result from the second stage of auto oxidation producing aldehydes and ketones due to peroxide oxidation (Lindsay, 1991) The rate of spoilage depends on several factors that include the fish species handling and storage conditions which are of major importance (Olafsdottir et al., 1997). Soon after harvesting or capture, fish must be refrigerated immediately in order to preserve fish quality. In addition to spoilage bacteria, it has been well documented that both fresh water and brackish water fishes can harbor human pathogenic bacteria particularly the coliform group. Fecal coliform s in fish are indicative of the level of pollution in their environment Proper handling, pretreatment and preservation techniques can improve the quality of fish and fish products and increase shelf life (Ghaly et a l. 2010) In Malawi, processing facilities at most beaches are poor and inad equate to meet rigorous sanitation s tandards Although smoking kilns of the I vory Coast type (Reynolds 1993) and chicken wire drying racks were built at some beaches for development demonstration projects in Mangochi, Salima, Nkhotakota and Zomba districts in the (FAO, 1993) open pi t roasting and reed drying racks are still the most prevalent ways of processing fish in Malawi The most common method of fish smoking in Malawi uses traditional smoking kilns and open pits wi th improvised wire gauze on top. Due to little or no refrigerat ion in many areas in Malawi antimicrobials that would aid in extending shelf life by at least two days would be most beneficial. The use of dried buffered vinegar powder has been documented to effectively enhance food safety in meat and poultry processing plants as well as enhance food quality stability and shelf life of various foods in different parts of the world.
14 Currently in Malawi, antimicrobial agents are not used to maintain fish quality, therefore, more losses result from microbial spoilage, and there are limited studies on the use of preservative agents for fresh fish in Malawi. The objectives of this research were to identify the existing knowledge gaps in fresh fish processing and handling methods among Malawian fish traders by administering a survey (Phase 1), and to determine the antimicrobial and sensory effects of dried buffered white vinegar powder o n storage stability of fresh Tilapia (Phase 2).
15 CHAPTER 2 LITERATURE REVIEW Geographical Location and Fishing Water Bodies in Malawi Malawi is a landlocked coun try with a total area of 118,48 4 km 2 of which about 20 % (24,405 km 2 ) is covered by water supporting over 800 species of fish. The largest water body in the country is Lake Malawi with an estimated surface area of 29,000 km 2 and a leng th of about 700 k m (FAO 2005 ) Other water bodies important for fishing activities include Lakes Malombe, Chilwa, Chiuta and six major rivers including : Shire, Linthipe, Bua, Dwangwa, Rukuru and Songwe. These water bodies s upport fisheries resources that are of social and economic importance to the country (FAO, 2005; Government of Malawi and GTZ, 2007). The Fishing Industry in Malawi The population of Malawi was reported to be 1 3 million in 2008 ( National Statistical Office 2008 ) with more than 65 % of the population living below the poverty line (i.e less than a dollar a day). Fish provides the most affordable source of dietary animal protein in Malawi and there is an increased demand for fish because of high population growth (National Statistical Off ice 2008 ) and the depleti on of fisheries resources due to overfishing These factors result in an inability of fishermen to meet the existing fish demand. Fish provides over 60% of the dietary animal protein intake of Malawians and 40% of the total protei n supply (Press Corporation Limited, 2007). Much of the fish is consumed in rural areas thereby contributing significantly to daily nutritional requirements of the poor rural population The sector is a source of employment, directly employing about 50,000 fishermen and indirectly about 350,000 people who are
16 involved in fish processing, fish marketing, net making, boat building and engine repair (Government of Malawi, 2009). Fish is a source of income that generates beach price local reven ue of 2.6 billion Malawi Kwacha s (US $24 million) annually. The contribution of fish to the Gross Domestic Product (GDP) was estimated at 4% (FAO 20 05) This is a significant contribution when compared with livestock which contributes 8% of the total GDP (Gover nment of Ma lawi and GTZ, 2007) T he existence of over 800 native fish species in Lake Malawi has also created ecotourism and an export trade for aquarium fish. The main fish resources are in inland lakes and a quaculture. Capture fisheries is practiced in all of the inland lakes which include Malombe, Chilwa, Chiuta, and two sections of th e Shire River (upper and lower S hire), with Lake Malawi as the main fishing ground. The Lake Malawi fisheries are multi species fisheries with distinct fisheries named after fish spe cies or fishing gears (Government of Malawi, 2009). The leading fishery in terms of commercial importance is the Chambo fishery, which consists of three Oreochromis species namely; Oreochromis karongae, Oreochromis lidole, and Oreochromis squamipinnis Oth er commercially important fish species include Haplochromis species Engraulicypris sardella, Copadichromis species, Bargrus meridionalis and Clariid gariepinus (Government of Malawi and GTZ, 2007). The typical fishing gear range s from pair trawlers, ring nets to less sophisticated gear that includ e beach seines, gill nets and fish traps. Haplochromines dominate the beach seine net catches, whereas, Engraulicypris sardella and Copadichromis species dominate the open water seine net catches. Oreochromis spe cies, Bargrus meridionalis
1 7 and Clariius gariepinus are either caught by gillnets or long lines (Government of Malawi, 2009) Fish production from aquaculture in Malawi is carried out on a small scale, with esti mated annual catches of 800 metric ton s (FAO, 20 05) and provides 2% of the households involved in fish farming (Russell et al., 2008). Types of Fisheries in Malawi The Malawi fisheries sector is classified into the small scale commercial sector, which is often called traditional, or the artisanal sector, and the large scale commercial sector with large capital investment. Except for effort limitations in the latter, the fishery operates on an open access basis, w ith entry allowed with payment of license fees. Although Lake Malawi is shared with bordering countries, there is no formal cross border fishing (Government of Malawi and GTZ, 2007) Small s cale c ommercial s ector The small scale commercial sector employs m any different types of g ear consisting of beach seines, open w ater seine nets, fish traps, gillnets hand lines and long lines. Dugout canoes and plank boats, with or without outboard engines, are the main fishing vessels. Fish landings average about 45,00 0 times per year, comprising predominantly small and large cichlids, Engraulicypris sardella and catfish However, there is a generally decreasing trend in annual catches of this sector (FAO, 2005). Large s cale c ommercial s ector The large scale commercia l sector is characterized by being highly mechanized and capital intensive. The fishery consists largely of pair trawlers (wooden boats about 8 m long, with a 20 40 horsepower inboard engine) and stern trawlers powered by
18 engines of 90 385 horsepower. All these are confined to the southern part of Lake Malawi which is relatively shallow, and is the most productive area Th is sector lands about 5,600 metric ton s per year of predominantly small cichlids ( Lethrinops and Copadichromis spp .) that represent appro ximately 21% of the total annual fish landings from Lake Malawi. Production has decreas ed because of the overexploit ation of the Oreochromis spp (Chambo) stocks (FAO, 2005; Government of Malawi and GTZ, 2007). Maldeco Fisheries is the largest commercial fishing and processing company in Malawi and fishes using three stern trawlers. Maldeco's annual catch amounts to over 70% of the total commercial catch and about 7% of the total Lake Malawi catch, which fluctuates between 26,000 tons and 47,000 tons annua lly. Maldeco operates a fish processing plant, which has freezing and cold storage facilities and three ice plants. Maldeco's main product is fresh fish representing over 90% of sales with only 10% processed including smoked, frozen gutted and frozen fille ts. Maldeco's name is synonymous with quality because the company's fish is always fresh cooled with ice and deliv ered straight from the trawler. The company operates shops in the Nchalo, Kasungu, Lilongwe, Zomba, and Blantyre districts and the shops are serviced using refrigerated trucks. The shops sell both retail and wholesale. The major wholesale customers are vendors who buy for re sale elsewhere (Press Corporation Limited, 2007) Fish Marketing in Malawi Fish Trading Fish trading in the artisanal se ctor of the fishery involves many people at different stages. Fish may change ownership many times, before they arrive at the consumer.
19 For most traders this allows great flexibility in their marketing activities. In terms of target species, the Oreochromi s species are considered to be the most valuable. S mall fish harvested in nets as by catch are also an impo rtant part of the fishery and are consumed by a large sector of the population So because every fish species has cu stomer s (FAO 1993). Higher income consumers usually buy fresh Oreochromis species from supermarkets and will only look for fish at produce markets if the fish is not available at the supermarkets. People with low incomes buy fish from the markets and choo se the best on a given day at a price they can afford (Russell et al., 2008). Imported Fish and Fish Products Fish imports are generally increasing from Zimbabwe, South Africa, Tanzania, Mozambique, Thailand and Namibia. The country imports fishmeal for livestock/poultry feed manufacturing companies (Government of Malawi and GTZ, 2007). Sun dried Kapenta ( Limnothrissa noidone and Stollothrissa tanganica ) is one of the major imported fish from neighboring countries; mainly Zambia.This fish is somewhat simi lar to Engraulicypris sardella Fish Processing in Malawi Fresh Fish As earlier stated, the fishing vessels consist of wooden boats, without freezing or chilling facilities for fish quality preservation. Ice exists in only two districts along the entire l ake (Mangochi and Salima). Furthermore, most fishing vessels in use are too small to carry ice on board, therefore, when the fish is caught it spoils easily Thus, high quality raw material for processing or preservation is lacking from the outset (FAO 19 93).
20 Ice is used primarily by middlemen who transport fish from the main fishing grounds to the markets. Ice blocks are placed in between fish layers in bamboo baskets, aluminum tins or cooler boxes. However, due to limited access to electricity, the use o f ice is limited. Maldeco Fisheries Limited is the only company that has shore based facilities, ice plants and chill storage facilities with a fish marketing and distribution n etwork throughout the country. The company supplies fish to both rural and urba n markets (Press Cooperation, 2007). In the northern region (Mzuzu City) catfish is gutted, cut into pieces and sold fresh at the city market. This only applies to bigger catfish with a lot of flesh (meat). No additives are added to the fish. Ice is used to keep the fish fresh before being sold; however, this depends on the availability of ice. In cases where ice is not available, fish spoilage occurs. Further Processed Fish In addition to preservation, fish can be further processed into different types of products to increase their market value. An example of value addition is the production of surimi and surimi based products. Surimi is a mechanically deboned, washed (bleached) and stabilized fish flesh. It is an intermediate product used in the preparati on of a variety of ready to eat food such as K amaboko, fish sausage, fish b a lls, fish burgers, fish fingers, fish cutlets a nd imitation shrimp products. The Surimi industry has grown in many countries worldwide, due to the emergence of Japanese restaurants and culinary traditions in North America, Europe and elsewhere ( Medina and Garrote, 2002 ) In Malawi further fish processing into prod u cts like these has not started Processing methods currently being practiced in Malawi include sun drying which is most ly used for small fish, (Utaka, Usipa and Kambuzi species) and smoking is used
21 on large sized species such as Clarius and Oreochromis species Other processing methods include par boiling for Engraulicypris sardella and pan roasting for Copadichromis Su ndried Fish Sun drying is done on open drying racks at the beaches. This is mostly done for small sized fish species e.g. Lethrinops Copadichromis spp and Engraulicypris sardella. Small sized fish are usually dried without gutting while bigger fish ( Cla rius and Oreochromis species ) are gutted before sun drying. Drying period ranges from four to seven days. Dried fish is packed in sacks or bamboo baskets and stored at room temperature (around 25 C ) while smoked fish is packed in bamboo baskets and stored at room temperature. Smoked Fish Shelf life extension of smoked fish as compared to fresh fish is due to the combination of low wat er activity and bactericidal and antioxidant components of wood smoke. In addition to shelf life extension, smoking is also used for flavoring and coloring (Hall 1997). In Malawi, fish smoking is considered as one way of prolonging shelf life of fish and it is mostly done for large sized fish species such as the O reochromis species and catfish. For smoking, fish are gutted, w ashed with water, placed in smoking kilns or on smoking racks and exposed to firewood smoke and heat. The smoking process results in non ready to eat, semi cooked smoked fish, which require s some form of cooking by the consumer before consumption.
22 Parboi led Fish This method is only used for Engraulicypris sardella Fresh fish is placed in hot water for a f ew minutes and rapidly cooled. The fish is then sun dried by layering on bamboo racks layered with mosquito nets to provide a smooth surface and avoid f ish breakage. The duration of sun drying may range from 5 to 10 days depending on sunlight intensity and overall atmospheric pressure. After drying, the fish is put in sacks and transported to rural and urban markets where it is sold. No packaging is done; instead, fish is sold on open boards and racks, Pan Roasting Pan roasting is used for small sized fish whereby fish are put on roasting pans plac ed above direct firewood heat. Pan roasted fish is intended to be sold within 2 to 3 days, however if the f ish is not sold after the three or four days, sun drying follows. In all the methods discussed (drying, smoking and pan roasting), preservative agents such as salt and natural or synthetic anti microbial agents are not applied to the fish. Microbiology of Fresh Fish The muscles of healthy fish are sterile and microorganisms reside at the surfaces such as skin, gills, and gastrointestinal tract of finfish (Gram, 2010). The number of microorganism s varies depending on the area of catch H owever, the skin typ ically contains 10 4 CFU /cm 2 the gills 10 6 CFU /g, and the digestive tract up to 10 8 CFU/g (Austin, 2002). The number of microorganisms in the digestive tract may vary from 10 4 to 10 9 CFU/g (Spanggaard et al., 2000). The presence of human pathogenic bacter ia such as Salmonella, Listeria monocytogenes, E.coli and toxin producing bacteria including Staphylococcus aureus, Clostridium botulinum and Bacillus cereus may not result in spoilage but in
23 microbiologicall y unsafe food and food products (Doyle and Beuch at, 2007). According to H uss 19 88 indigenous pathogenic bacteria of fish include Vibrio spp ., Clostridium botulinum (Type E), Listeria monocytogenes Aeromonas spp ., and Shewanella algae Muscle food can become contaminated during the harvesting process f rom the environment or from direct contact with the intestinal tract contents. Contamination can result in illness if the processor or consumer does not handle the product appropriately (Harris et al. 2006). The presence of coliforms, especially fecal col iforms and E. coli are indicative of fecal contaminati on in the water or post harvest. Certain E.coli strains Enteropathogenic EPEC Enteroinvasive EIEC, Enterotoxigenic ETEC, and Enterohaemorrhagic EHEC are pathogenic and lead to serious human diseases ( D oyle and Beuchat 2007 ) Total plate counts are performed to give an estimate of the total microflora present on the fish and can be used as indicato r s of spoilage when numbers reach 10 8 to 10 9 CFU/g in fish. The spoilage microflora of fish which reach p opulations of 10 8 to 10 9 CFU/g after 2 to 4 weeks, is usually dominated by pseudomonads and shewanellae (Sperber and Doyle, 2009). The shelf life of fresh fish is limited by biochemical and microbiological changes. Microbial growth and metabolism is a major caus e of fish spoilage which is characterized by production of biogenic amines such as putrescine, histamine, and cad a verine, organic acids, sulphides, alcohols, aldehydes, and ketones, all of which produce unpleasant and unacc eptable off flavors and odor s. U nder aerobic conditions the shelf life of fresh P earlspot ( Etroplus suratensis Bloch ) is limited due to the rapid
24 growth of microorganisms such as Pseudomonas, S hewanella putrefaciens and A eromonas hydrophila ( Ravi Sankar et al., 2008 ). Al harbi and Uddin ( 2005) determined that Shewanella putrefaciens, Corynebacterium urealyticum Aeromonas hydrophila and Flavobacterium spp dominated the bacterial flora associated with the intestine of hybrid tilapia ( Oreochromis niloticus x Oreochromis aureus ) and S hewanella putrefaciens was the m ost dominant spoilage organism. However, spoilage bacteria var y with water type ( e.g. freshwater saltwater or brack ish, etc.) and fish type. The bacteria that subsequently become important in spoilage of fresh finfish are present only in small proportions at capture (Gram, 2010) Storage of finfish at ambient temperature leads to rapid growth of mesophilic Gram negative bacteria belonging to Vibrionaceae or Enterobacteriaceae (Len, 1987; Liston, 1992). These bacteria reduce trymethylamine oxide t o trymethylamine by the enzyme trymethylamine oxidase and produce several sulfides including hydrogen sulfide and dimethyl sulfide, resulting in short shelf life, typically less than 24 hours. Other compounds produced due to microbia l activity are indole, skatole, putrescine and cadaverine from proteins (Ashie et al ., 1996) which are also indicative of spo i lage The enzymes produced by spoilage micro flora also cause proteolysis, deamination, and decarboxylation resulting in accumulat ion of unpleasant metabolites and loss of flavor substances which leads to sensory rejection of the fish. Cooling, mostly in flaked or crushed ice, is the most common and most effective method for preservation of fresh finfish (Gram, 2010). Vogel et al. ( 2005) determined that the number of H 2 S producing bacteria in iced stored marine fish (cod, plaice and flounder) varied between 10 1 and 10 3 and constituted between 0.1 and 10% of the total
25 bacterial count. After 3 weeks of storage, the counts had increased to 10 8 to 10 9 and H 2 S producing bacteria constituted 2 to 30% of the viable count. In the warm summer months, several of the H 2 S producing bacteria were mesophilic Shewanella algae, which is a human pathogen. These bacteria quickly disappeared upon iced s torage and the psychrotrophic H 2 S producing bacteria subsequently dominated the spoilage bacterial community. Shelf life of raw fish depends on s to rage conditions, qualitative and quantitative composition of the initial microflora related to the environm ent al habitat of the fish and intrinsic factors (Gram and Huss, 2000 ). The se intrinsic factors are the inherent properties or parameters of fish muscle and they include moisture content, water activity (a w ), pH, oxidation reduction potential (Eh), and nutr ient content (Doyle and Beuchat 2007 ). Apart from these predisposing factors, spoilage bacteria can originate from gastrointestinal sources. Evisceration or gutting prior to storage can prolong the shelf life by reducing the microbial load of the spoilage microorganisms (Al Harbi and Uddin, 2005). Improvements in shelf life of fish and fish products can have an important economic benefit by reducing losses attributed to spoilage and by allowing the products to reach distant and new markets (Kykkidou et al. 2009 citing Rhodehamel, 1992). Need for Shelf Stable Processed and Packaged Fish and Fish Products Fishery Products (non processed fish) In Malawi, fish is commonly sold whole, fresh, and un packaged, or sun dried or gutted and smoked. However, on a ve ry small scale, fried and salted fish is also sold mostly as a snack for travelers at bus stops. Fish production from capture fisheries has declined drastically in recent years. This is evidenced by the low total fish landings and
26 the per capita consumptio n which has fallen from 13 k less than 5 k g/person/year in 2005. However, there is increased total fish demand due to high population growth and urbanization. In spite of the economic, social and nutritional benefits of the fi sheries sector, concerns have been expressed about the sustain ability of the fisheries sector due to dwindling supplies from capture fisheries (FAO 1993) In order to improve the livelihoods of fishermen, processors and traders as well as scale up the role played by the sector in developing countries, there is need to reduce the fish losses incurred from fish spoilage. The most obvious means of increasing supply of fish without increased landings is by reducing post harvest losses of wha t is presently caugh t (FAO, 2005 ). The post harvest sector of fish production in the country is still underdeveloped. This translates to losses estimated to be as high as 30% during some rainy seasons. Fish losses also result from inadequate processing and storage facilities, and fish distribution problems due to inadequate means o f transportation (FAO, 1993). Fresh fish without ice is only transported to adjacent lakeshore communities. For transportation most fish traders hitch a ride or travel by public transport, mainly b uses, trucks and pickups for long distances. For short distance, som e own bicycles (7%), motorcycles (1%) or pick up truck s (2%). Traders without transport are able to hire bicycles and helpers to transport the fish, usually in sacks, baskets or cartons, f rom the beach to the main road (FAO, 1993 ). Current Preservation Processes in Malawi Cooling and freezing are current preservation processes used in Malawi (Ghaly et al., 2010). No chemical approaches are used for preservation, although in 1982, the FAO C odex Alimentarius Committee approved use of actellic (Pirimiphos methyl) on
27 small species of fish ( Haplochromis and Lethrinops ). This chemical has successfully reduced losses in fresh fish due to blowfly larvae infestation, especially in the rainy season. The use of a ctellic on larger sized species of fish was not approved, as it was found that the thick muscles of these fish absorbed higher residues than the legal limit (5 mg/kg) (Hara and Mkoko, 1993). Historically, salting, drying, smoking, fermentation and canning were the methods for fish spoilage prevention and shelf life extension. In response to consumer demand for texture, appearance and taste, fish processing not only serves as a means of preservation, but it also brings value addition to the produ ct, as different types of products with a wide range of organoleptic qualities are developed. This also improves the market potential of the product (Ghaly et al., 2010). In Malawi, smoking is done as a means of preservation and value addition. In additio n to processing, packaging is used to protect products from conta mination and prevent spoilage. Packaging also extends shelf life of products, facilitates distribution and display of products, improves consumer appeal, and facilitates the display of inform ation on th e products (FAO, 1996). Currently s moked fish is not packag ed in Malawi Application of Antimicrobial Interventions for Control of Spoilage Bacteria in Tilapia Organic acids and their salts (acetic, lactic, propionic and sorbic) have been tradi tionally used as food preservatives and are generally recognized as safe substances (GRAS) They (organic acids) are approved as food additives by the US FDA, the E uropean Commission (now the EU) and the FAO/ WHO (Surekha and Reddy, 1999). Organic acids ha ve been successfully used to control spoilage and
28 pathogenic bacteria in meat (Hamby et al.1 987), poultry (Zeitoun and Debevere, 1990) and fish products. Harris et al. ( 20 06 ) reported that the application of 2% acetic acid resulted in 0.5 and 0.6 log reduc tion of E.coli 0157:H7 and Salmonella typhimurium in ground beef. Intermittent sprays of acetic acid resulted in significant reductions (1.8 4.3 log CFU /cm 3 ) of aerobic plate count s in vacuum packaged strip loin, boneless rib and clod stored at 2 o C for 28 days (Hamby, 1987). An application of 2% acetic acid reduced the incidence of Salmonella on pork cheek meat in addition to reducing aerobic plate and coliform count (Frederic k et al., 1994) Acetic acid is a strong inhibitor to the proliferation of microo rganisms and also inhibits most enzymatic activities which results in retardation of protease activity in fish muscle (Hoitsy et al., 2012). Sodium lactate, lactic acid and acetic acid have been used to retard growth of microo rganism s in fresh catfish fill ets (Williams et a l ., 1995), Florida mullet, and black sea bass fish (Williams and Rodrick 1990). Williams and Rodrick (1990) determined that although lactic acid and acetic acid at 0.5 and 1.0% retarded the growth of microorganisms, the fillets were di s c olored due to denaturation of the myoglobin pigment. Sodium lactate at 1.0 and 2.0% retarded the growth of bacteria without denaturation of the fish myoglobin. The antimicrobial activity of most organic acids is the result of a decrease in pH and a spec ific antimicrobial effect of the un dissociated molecule, particularly the dissociation constant (pKa) and the concentration of the acidulant used (Zeitoun and Debevere, 1990). Zeitoun and Debevere (1990) determined that treatment of poultry carcasses with 10% (w/v) lactic acid/sodium lactate buffer (pH 3.0) immediately after slaughter exerted antimicrobial effect s while maintaining a low pH in the skin during storage without apparent adverse effects on sensory quality. The shelf life of poultry
29 legs treat ed with 10% lactic acid buffer was extended to 12 days at 60 o C while untreated chicken legs had a shelf life of 6 days at the same temperature. Papadoupolos et al. (2 003) stored fresh aquacultured Sea B ass (Dicentrarchus labrax) in ice for 16 days and dete rmined that the shelf lif e of whole ungutted and gutted S ea B ass wa s 13 and 8 days, respectively. The researchers concluded that gutting the fish resulted in higher level s of spoilage micro organisms a nd shorter shelf life than the ungutted fish The use o f dried vinegar at different levels has effectively enhance d food safety (in meat and poultry processing plants) food quality and stability and enhanc ed the shelf life of various foods in different parts of the world. Currently in Malawi, antimicrobial a gents are not used to maintain fresh fish quality, therefore, more losses result from microbial spoilage, and there are limited studies on the use of preservative agents for fresh fish in Malawi.
30 CHAPTER 3 MATERIALS AND METHOD S Background to the study Fi sh processing industry development in Malawi is still in its infancy stages and lacks modern technology and equipment for shelf life extension and value addition Fifty percent of the fish is s undried and 30% is smoked (Russel et al., 2008) Currently, the use of ice for fresh fish is limited and no chemical additives or preservatives have been documented to be used on fresh fish in the country. Salt is not usually used in fish processing because it is not cost effective (Hara 1993). However, there is grow ing demand for fresh fish m ainly in urban/city markets. The hypothesis of this study was that the use of dried white buffered vinegar for ice stored fish in cooler boxes would inhibit the proliferation of the total bacteria population Gutting fish prior t o application of dried white buffered vinegar would reduce the initial m icrobial load and extend the shelf life of fresh f ish under ice storage in coolers Main objective The main objective was t o evaluate the effec ts of DV and gutting on the quality stabi lity and shelf life extension of fresh Malawian Tilapia ( Oreochromis species). Specific objectives To determine the antimicrobial effect of dv on shelf life extension of fresh Malawian tilapia ( Oreochromis species) under ice storage. To determine the senso ry characteristics associated with the use of the dv treatment.
31 The study was done in two phases. In phase 1 a survey was conducted to identify the existing knowledge gaps in fresh fish processing and handling methods among Malawian fish traders. Phase 2 involved investigations o f the antimicrobial and sensory e ffects of dried white buffered vinegar and gutting on storage stability of fresh Tilapia Phase 1. Survey on Current Methods Employed by Fish Traders in Lilongwe, Limbe and Blantyre City Markets f or Fresh Fish Quality P reservation in Coolers and the Effectiveness in Reducing Fresh Fish Spoilage Prior to evaluation of the dried vinegar product for antimicrobial properties on the Tilapia a survey was conducted to determine current methods employed by fish traders in three selected city markets (Lilongwe, Limbe and Blantyre) to preserve fresh fish harvested from Lake Malawi. The survey was designed to identify the existing knowledge gaps in issues related to fresh fish spoilage and preservation, to d etermine of food sa fety and fish handling issues, to determine existing methods fo r fresh fish quality preservation used by fresh fish traders, and to establish the effectiveness of those methods in prolonging the shelf life of fresh fish (Appendix A) The survey was conducted in three city markets that included Lilongwe city market in Lilongwe Blantyre city market and Limbe city market in Blantyre. Lilongwe and Blantyre were selected because they are the main cities in Malawi. Malawi has four cities which include Blantyre Lilongwe Zomba and Mzuzu Zomba and Mzuzu are growing cities. The survey was conducted face to face with the participants using a responses were recorded on the questionnaire. Eighteen questionnaires were
32 administered with six questionn aires used per city market. Respondents were randomly selected and they participate d voluntarily. Convenient meeting times were scheduled with each participant separately. Phase 2. Evaluation of Dried Buffered Vinegar for Antimicrobial and Sensory Charac teristics on Whole Un gutted and Gutted Malawian Tilapia Fish Fresh Fish Harvesting, Treatment and Evaluation Fresh Tilapia fish ( Oreochromis species ) harvested from Lake Malawi, and was purchased f rom Maldeco Fisheries Limited. The fish was transported t o the Microbiology lab at Chancellor College at the University of Malawi for treatment application, further storage and analysis. Upon arrival at the lab, dried buffered white vinega r powder (DV, WTI, Inc., Jefferson, GA ,U.S.A) was reconstituted in tap wa ter to produce 0, 5.0, 8.0 and 10.0% (w/v) DV treatment solutions T he fish were divided into two equal batches Fish in batch no. 1 w ere left whole ungutted and treated with 0 5 .0 8 .0 or 10 .0 % (w/v) DV solutions by spraying to completely drench the fish The fish was allowed to drain for 10 seconds on a presterilized wire rack and placed in layers of ice in 36 30 22 cm cooler boxes Fi sh in batch no. 2 were gutted with the head and gills left intact and treated in the same manner as described for bat ch no. 1 All fish w ere gutted using clean sanitized knives in a manner that avoid ed cutting the intestinal tract. The gills were left intact to evaluate gill color. The cooler boxes were assigned letters for identification where coolers A, K, W, and M we re used for un gutted whole fish and coolers D, G, Q, and J were used for gutted whole fish. Each cooler box contained an equal number of fish layers. Initially, the bottom of each cooler was covered with a layer of ice, followed by a layer of fish. This process was continued u n til the cooler was filled to capacity. The top layer inside
33 each cooler was ice. The thickness of ice layers was standardized at approximately 15 centimeters. The experiment was repeated two times. Fish internal temperature was reco rded after every 24 hour period and the ice was replenished. A thermometer was inserted into one fish from each layer in the cooler and the average inte rnal temperature was recorded. The fish coolers were stored at 25 C and analyzed after 0, 3 and 5 days for sensory characteristics, and after 0, 1, 3, and 5 days of storage for microbiology (aerobic plate count, total coliforms and generic Escherichia coli ) and pH. Microbiological Analyses The official AOAC ( 2000) method for Aerobic Plate Count using Petri film (Petrifilm Aerobic Plate Count 3M Microbiology St. Paul, MN USA) and 991.14 method for Coliforms and Escherichia coli Counts using E.coli and Total Coliform Petrifilm Plate s were used for microbial analysis. Duplicate 25 gram samples per treatment were collected from 2 randomly selected fish per cooler box The samples were collected by cutting the flesh below the dorsal fin using sterilized knife and forceps. After cutting, 25g of the cut f ish meat was weighed and placed in sterile stomacher bags. The 25 gram samples were combined with 225 ml of 0.1% sterile Peptone water (Beckon Dickinson and Company. Sparks, MD, U.S.A). To suspend bacterial load, t he samples were hand massaged for 1 minute in the sterile stomacher bags (Stomacher lab system, Sewar d Limited, U.K). Serial dilutions of 10 1 to 10 6 were prepared, and 100l aliquots of each were pipetted onto pre hardened Tryptic Soy Agar (TSA) for total aerobic plate count (APC s ). One ml aliquots of the sample homogenate were pipetted onto 3M T otal C oliform Petrifilm and E scherichia c oli Petrifilm (Petrifilm TM St. Paul, MN, U.S.A.) for
34 total coliforms and generic E. coli respectively All plates were incubated at 35 1 C. The bacterial colonies on plates were counted, recorded and averaged. Sensor y A nalysis A sensory panel consisting of 6 members was trained to conduct sensory evaluation and evaluation of quality attributes (skin, exterior odor, gills, consistency flesh color and eyes) on the Tilapia Q uality attribute ratings were adopted from Ca mpos et al. ( 200 5 ) with some modifications for the species used in this study ( Appendix C ). The fresh fish attributes were rated using a four point quality scale where 4 = highest quality, 3 = good quality, 2 = fair quality, and 1 = unacceptable ( Appendix D ). Statistical Analysis The data w ere analyzed in a randomized block design. A 2 x 4 x 4 x 2 factorial treatment arrangement with two levels of fish (who le un gutted and whole gutted), four levels of DV (0%, 5.0%, 8.0% and 10.0%), and four storage days (0 1, 3, and 5 days) (Appendix E ) with two replications was employed A total of 64 samples were analyzed for the microbiology and sensory evaluation. Data were analyzed using SAS General Linear Model procedure (SAS Institute, 2002). The model included the main effects of DV treatments, gutting, storage days, an d treatment by day interaction. Comparisons among means were performed using SAS Duncan Multiple Range test. Treatment effects and differences were considered significant when P < 0.05. T he face to face survey questionnaire w as completed by 18 participants that included 6 participants per 3 city markets. A total of 30 responses were solicited from each participant on the questionnaire. The data were analyzed using SPSS program to generate frequencies and percentages for the responses.
35 CHAPTER 4 RESULTS AND DISCUSSI ON Phase 1. Results of Survey on Current Methods Employed by Fish Traders in Lilongwe, Limbe and Blantyre City Markets for Fresh Fish Quality P reservation in Coolers and the Effectiveness in Reducing Fresh Fish Spoilage Introduction to Survey Difficulties were experienced during the solicitation of participants for the survey. G eneral reluctance and unwillingness to participate were the main challenges encountered because of the following reasons given by the respondents: C onsequence s of this survey would result in an increase in market fees paid to the Malawi Revenue Authority. There we re n o meaningful benefits from previous surveys. Because the potential respondents do not follow proper s elling procedures (i.e., selling of spoiled fish), t hey were afraid that the survey was being conducted by the Malawi Bureau of S tandards. There was no economic incentive in the form o f cash for their participation. Accompanied with these factors was lac k of willingness of the respondents to answer questions 1 through 1 0 14 through 16 and 20 (Appendix A) As a result, there were missing data for some of the questions. Survey O utcomes The age categories of the par ticipants were 23 27 years (28%), 30 39 years (50 %), 40 45 years (16 %) and 54 years (6 %) (Partial response for Question 1) Three fish species that included Oreochromis species locally known as C hambo and Tilapia Engraulicypris sardella locally known as Usipa and Rhamphochromis species locally known as M cheni were report ed as the most prefered species for sale by the fish
36 traders (Question 11) Among the three species 83% of the respondents preferred Tilapia (Chambo) for its great taste while all three species had an overall score of 33% fo r great taste preference. Engraulicypris sardella ranked high for cheap producer price (50%) and high market value (50%) (Question 12) The type of customers that their fish was sold to were primarily other traders, consumers and processors. Most of the r espondents (67%) declared that consumers were their preferred customers followed by other traders (17%) and consumers and processors (11%) (Question 13) The results indicate d that consumers are the preferred customers. The condition of fish at the time of sale can have a direct impact on the sales. It was evident that there wa s a general lack of consumer awareness o f fish quality standards, because consumers buy fish regardless of the condition of the fish from a quality and safety point of view. In additi on, consumers were preferred by most traders because most consumers can only afford to buy fish at a reduced price or cheaper price when fish shows signs of spoilage. A higher percentage of respondents (44%) reported that they reduc ed the selling price whe n fish shows signs of spoilage Approximately 17% of the respondents reported that they would sell fish showing signs of spoilage to traders and 6% would resell to further processing operations. In order to avoid or retard fresh fish spoilage, the use of i ce in coolers was reported by 6 1 % followed by rinsing fish in clean water (28%) while gutting and use of other methods such as sun drying had the lowest rating (5.6%) ( Question 17) The survey revealed that sun drying and smoking methods used by fish tra ders when fish show signs of spoilage are preferred by the traders as a way of masking the obvious signs of spoilage from consumers.
37 Major challenges experienced by the fish traders were lack of access to fresh fish shelf life extension methods and lack o f knowledge on approved fresh fish quality preservatives such as vinegar (Question 18) Sixty one percent of respondents u se d bamboo baskets as containers for carrying fresh fish during transportation 22% used both cooler boxes and bamboo baskets and 1 1% use cooler boxes only (Question 19) These local packaging materials compromise the quality of fresh fish during transportation. Ninety five percent of the respondents used less than 500 g of ice per kilogram of fish as compared to 5% of the respondents who used of more than 500 g of ice per kilogram of fish (Question 21). In regard to fresh fish preservation methods 22% of the respondents declared that fish spoils after 24 hours ( 1 day ) and 16 % of the respondents declared that fish spoils after 48 hour s ( 2 days ) A higher percentage of the respondents (about 54%) declared that fish spoils after a maximum period of 8 hours (Question 22) Twenty two percent of the respondents described signs of fish spoilage as bad smell, fish scales falling off and muscl e tenderness 11% reported spoilage as b ad smell sunken eyes gill discoloration and presence of flies and 22% described spoilage as bad smell sunken eyes and gill discoloration (Question 23). As alternative actions f or decreasing fresh fish spoilage in their business, 44% of the respondents cited the reduction of selling prices as the best action to avoid losses. Seventeen percent of the respondents suggested re sale of fish to other processors as the best action when fish begin s to show signs of spoila ge and 6% of the respondents
38 mentioned further processing of fish by frying or sun drying as the best actions taken when fish starts showing signs of spoilage (Question 24) In regard to extension services and training, 95% of the respondents had n o acces s to any extension services and 100% had no formal training in food safety, fresh fish quality control, fish handling and basic personal hygiene (Questions 25, 26, 27 and 28) The findings of this survey revealed that there is a general lack of standard fish quality preservation methods used by fish traders in the Malawi city markets (Question 30). It was also revealed that t here is a significant knowledge gap among the fish traders in fresh fish basic handling and quality preservation methods. These fact ors coupled with poor or lack of access to facilities and technologies for fresh fish quality preservation have resulted in a n extremely high fish spoilage rate. Phase 2. Evaluation of Dried Buffered Vinegar for Antimicrobial Properties and Sensory Charact eristics on Whole Un gutted and Gutted Malawian Tilapia Fish Microbiological Analyses Aerobic plate c ount The 10% DV treatment had lower (P < 0.05) APC than the control on all storage days for the ungutted fish (Table 4 1) The 5 and 8% DV treatments for the ungutted fish were also lower (P < 0.05) than the control on d 3 and 5 of storage. The 10% D V treatment for the gutted fish resulted in lower (P < 0.05) APC when compared to the control on d 0 and 5. All DV treatments for the gutted fish had lower APC on d 5 when compared to the control The DV treatments for gutted and ungutted fish resulted in APC counts of 7.00 log CFU /g or lower after 5 days storage while the ungutted and
39 gutted control s had counts of 9 log CFU/g Th is observation revealed that the DV treatments retarded the growth of APC counts through d 5 of storage on ice Barnes (1976) and Cunningham (1979) reported that meat spoils when bacteria counts reach 8 log CFU /g The ICMSF ( 2005 ) reported APC counts of 10 5 CFU/g as standard for fresh f ish, and 10 6 or higher for fish as it begins to spoil (Yousef Ali et al., 2007). W hen gutted fish and ungutted fish were compared by day and treatment (Tabl e 4 2 ), the data demonstrated that gutting did not affect (P > 0.05) APC for 0, 5 and 8% DV treatmen ts on d 1, and 5 of storage. However, gutting decreased APC on d 3 for the 10% DV rate. Total coliform c ounts Total coliform counts increased (P > 0.05) as storage time increased fro m d 0 to d 5 of storage for ungutted and gutted fish (Table 4 3 ) All TCC were less than 2.50 log CFU/g for all treatme nts Gu tted fish had lower counts on d0 at the 10% DV rate while ungutted fish had higher counts at the same DV rate This trend was reversed on d1 and d3 where ungutted fish had lower counts than gutted fish f or the 8% DV rate. The ICMSF reported TCC of less than 100 CFU/g as standard for fresh fish (Yousef Ali et al., 2007). Of more significance than TCC are fecal coliforms and E. coli in fresh fish which are indicators of fecal contamination due to bad water or post harv est handling and poor hygiene. E. coli is found predominantly in the gastrointestinal tract of humans. Generic E. coli counts Significant interaction (P < 0.05) was revealed between DV treatment and storage time, which was due to increase in E. coli counts as storage time increased. The 10% DV treatment resulted in lower (P < 0.05) E. coli counts for the ungutted and gutted fish
40 through 5 days of storage when compared to the respective control s (Table 4 4) The 5% and 8% DV treatments resulted i n lower E. coli counts on d 0 and d 5; respectively for the ungutted fish. The 8% and 10 % DV treatment was also effective in reducing E. coli on d1 for gutted fish when compared to the control. While on d 3, only 10% DV had significantly lower counts than the other DV rates and control. On d 5, a similar tren d was observed where 8% and 10 % DV rates had significantly lower counts when compared with the control. In general it was observed that an increase in the DV concentration resulted in a general decreas e in the E coli counts. The Seafood Network Information Center (2007) reported standards for E.coli in fresh fish as 11 CFU/g ( 1 (2.70 log CFU/g) for marginally acceptable quality The E. coli counts in this study for ungutted fish ranged from 0.45 log CFU/g (10% DV treatment on Day 0) to 2.77 log CFU/g (8% DV treatment on Day 5) for DV treatments and 1.74 log CFU/g to 4.78 log CFU/g for the controls F or gutted fish E. coli counts ranged from 0 .93 log CFU/g (10% DV treatment on Day 1) to 3.08 log CFU/g (5% DV treatment on Day 5) for DV treatments and 1.99 log CFU/g to 3.19 log CFU/g for the controls The data demonstrated that all ungutted fish treated with DV were in the good quality r ange on Days 0 1 and 3. On Day 5, except for the 10% DV treatment all treatments were considered l ess than marginally acceptable Counts for the ungutted controls were in the good quality range on days 0 and 1, b ut decreased to less than mar g i nally acceptable on days 3 and 5. As for the gutted fish, all fish treated with 8 and 10% DV were in the range of good quality on all storage days. Except for Day 0 for the
41 controls, all control samples and samples treated with 5% DV were below marginally acceptable The control on Day 0 had good quality Sensory Evaluation of Fresh Whole, gutted Oreochromis species Sensory Panels for Cooked Fish A total of 6 panelists were trained for evaluation of the cooked fish. However, on the evaluation days, pane lists were unwilli ng to taste the cooked samples. Therefore an informal evaluation of the cooked fish was conducted by 4 untrained individu als. T he panelists did not detect off flavor or off odor in the treated and control samples on day 5 of evaluation f or ungutted and gutted fish. The general comments were that all ( No quantitative data were recorded) Sensory Evaluation of Quality Attributes for Uncooked Oreochromis species The sensory evaluation for t he uncooked whole ungutted and gutted fish is presented in Table 4 6 for the quality attributes skin appearance odor, gill appearance flesh color and eye s Skin The panelists rated the skin of all ungutted and gutted fish in all treatments in the range o f good quality (3.67) to hi ghest quality (4.00) on Day 0. As storage time increased to 3 and 5 days, the skin began to deteriorate. The fish was rated unacceptable for the un gutted control on Day 5, and unacceptable for the gutted 5% DV treatment on Days 3 and 5. On day 5, except for the 5% DV treatment for the gutted fish, all DV treatments, ungutted and gutted, were in the range of good (8% DV treatment for gutted only) to fair which suggested that the DV treatments maintained fair skin quality through 5 days storage.
42 Odor The panelists rated the odor of all ungutted and gutted fish in all treatments in the range of good quality (3.67) to highest quality (4.00) on d 0. T he 8 and 10% DV treatments resulted in fish with fair to high qualit y odor through 5 days storage. The 0 and 5% DV treatments resulted in fair to unacceptable quality afte r 3 and through 5 days storage. This would imply that higher levels of DV (8% and 10%) had a more pronounced effect on the microbial activities associated with the p roduc tion of the odor than 5% and the control This indicated that the application of DV was effective in limiting some of the microbial activity to a certain extent. Gills The panelists rated the color of the gills of all ungutted and gutted fish in all treatm ents in the range of good quality (3.67) to highest quality (4.00) on Day 0. Appearance of the gills decreased as storage time increased, a lthough not always significant. Except for the control, the panelists rated the appearance of the gills for the ungut ted fish fair to good quality on 3 through 5 storage days Similar trend was observed for the appearance of the gutted fish. Except for the control and 5% DV treatment on Day 5, the panelists rated the appearance of the gills for the gutted fish fair to go od quality on Days 3 through 5. Flesh color The panelists rated the flesh color of all ungutted and gutted fish in all treatments in the range of good quality (3.67) to highest quality (4.00 10% DV treatment on Day 0 for gutted fish ) on Day 0. The paneli sts reported deterioration in the control and 5% DV treatment for gutted fish on Days 3 and 5 as unacce ptable. The 8 and 10% DV
43 treatments resulted in ungutted and gutted fish with ratings of fair to high quality on all storage days. E yes The panelists ra ted the eyes of all ungutted and gutted fish in all treatments in the range of good quality (3.67 ) to highest quality (4.00) on d ay 0. The 8 and 10% DV treatment s resulted in fair to good quality scor es (P > 0.05) for fish eyes on d ays 3 and 5 (except gutt ed, Day 5) The control d eteriorated to unacceptable on d ays 3 and 5 for the ungutte d and gutted controls. Deterioration to unacceptable quality was a l so observed for 5% DV treatment for ungutted fish on d ay 5 and gutted fish on d ays 3 and 5. pH The applic ation of DV did not affect the pH of the fish for the gutted and un gutted whole Tilapia ( P > 0.05). A ll pH values ranged from 7.0 to 7.2 for all treatments (Table 4 5) Very small amounts of lac tic acid are produced post mortem combined with very high pH of >6 of fish (Gram and Huss, 1996) This has important consequences for the microbiology of flesh fish as amongst other factors it allows pH sensitive bacteria Shewanella putrefaciens to gro w. These results suggest that DV treatmen t did not negatively alt er the pH of fish but maintained it. Therefore the application of DV did not result in adverse effects on the sensory attributes (production of a tangy taste associated with low pH) of fish.
44 Table 4 1 Effects of dried buffered vinegar and gutting on aerob ic plate counts of fresh Malawian Tilapia ( Oreochromis species ) stored on ice in cooler boxes for 5 days Gutting DV 1 (w/v % ) Day 0 Day 1 Day 3 Day 5 SEM Ungutted whole fish 0% 6.20 az 6.28 az 7.96 ay 9.32 ax 0.13 5% 5.46 abz 5.67 az 5.65 by 6.67 bx 0.1 3 8% 5.38 abz 5.47 abz 5.99 by 6.87 bx 0.13 10% 4.64 bz 4.65 bz 5.28 by 5.74 cx 0.13 SEM 0.13 0.13 0.13 0.13 Gutted whole fish 0% 5.63 az 6.57 az 6.70 ay 9.29 ax 0.13 5% 5.92 az 6.15 az 6.44 ay 7.04 bx 0.13 8% 5.53 az 5.63 az 6.04 ay 6.93 bx 0.13 10% 4.7 0 bz 6.31 az 6.44 ay 6.59 bx 0.13 SEM 0.13 0.13 0.13 0.13 1 DV = Dried white buffered vinegar a c means in the same column with the different superscript are significantly (P < 0.05) X Z means in the same row with different superscript are significantly d ifferent (P < 0.05) SEM means standard error of the mean
45 Table 4 2 Effect of g utting on APC of f resh Malawian Tilapia ( Oreochromis species ) treated with dried buffered vinegar and s tored on ice in cooler boxes for 5 days Gutting Day 0 Day 1 Day 3 Day 5 DV 1 (w/v %) SEM 0% Gutted 5.63 ax 6.57 ay 6.70 az 9.29 az 0.13 0% Un gutted 6.20 ax 6.28 ay 7.96 bz 9.32 az 0.13 5% Gutted 5.92 ax 6.15 ay 6.44 az 7.04 az 0.13 5% Un gutted 5.46 ax 5.67 ay 5.65 az 6.67 az 0.13 8% Gutted 5.53 ax 5.63 ay 6.04 az 6 .93 az 0.13 8% Un gutted 5.38 ax 5.47 ay 5.99 az 6.87 az 0.13 10% Gutted 4.70 ax 6.31 ay 6.44 az 6.59 az 0.13 10% Un gutted 4.64 ax 4.65 by 5.28 bz 5.74 bz 0.13 1 DV = Dried white buffered vinegar a b means in the same column with the different superscript are significantly different (P <0.05) x z means in the same row with different superscript are significantly different (P < 0.05) SEM means standard error of the mean
46 Table 4 3 Effect of DV on Total coliforms of gutted and u ngutted f resh Malawian Tilapi a ( Oreochromis species ) stored on ice in cooler boxes for 5 d ays Gutting DV 1 (w/v) % Day 0 Day 1 Day 3 Day5 Ungutted SEM 0% 0.80 a x 1.78 a x 2.06 a x 2.32 a x 0.02 5% 0.59 a x 1.17 a x 1.88 ab x 1.95 a x 0.02 8% 0.15 a x 0.65 b x 1.09 b x 1.78 a x 0.02 10% 0.54 a x 0 .93 a x 1.27 ab x 1.93 a x 0.02 SEM 0.02 0.02 0.02 0.02 Gutted 0% 1.21 a x 2.04 a x 2.32 a x 2.46 a x 0.02 5% 1.18 ab x 1.50 ab x 1.75 a x 2.16 a x 0.02 8% 1.08 ab x 1.38 ab x 1.63 a x 2.11 a x 0.02 10% 0.45 b x 1.13 b x 1.50 a x 1.64 a x 0.02 SEM 0.02 0.02 0.02 0.02 1 DV = Dried white buffered vinegar a c means in the same column with the different superscript are significantly different (P <0.05) x z means in the same row with different superscript are significantly different (P < 0.05) SEM means standard error of the mean.
47 Table 4 4 Effect of gutting and dried buffered vinegar treatment on E. coli of fresh Malawian Tilapia ( Oreochromis species) stored on ice in cooler boxes for 5 days Gutting DV 1 (w/v %) Day 0 Day 1 Day 3 Day 5 Ungutted 0% 1.74 abx 2.63 ay 3.12 az 4.78 az 0.09 5% 2.38 ax 2.24 ay 2.65 az 2.74 bz 0.09 8% 1.47 bx 1.72 aby 2.62 az 2.77 bz 0.09 10% 0.45 cx 1.35 by 1.38 bz 2.22 bz 0.09 SEM 0.09 0.09 0.091 0.09 Gutted SEM 0% 1.99 abx 2.79 ay 3.09 az 3.19 az 0.09 5% 2.71 ax 2.72 ay 3.02 az 3.08 az 0.09 8% 1.71 b x 1.33 by 2.44 az 2.45 abz 0.09 10% 1.24 bx 0.93 by 0.94 bz 1.67 bz 0.09 SEM 0.09 0.09 0.09 0.09 1 DV = Dried white buffered vinegar a c means in the same column with the different superscript are significantly different (P<0.05) x z means in the s ame row with different superscript are significantly different (P < 0.05) SEM means standard error of the mean
48 Table 4 5 Effect of DV on pH of g utted and u ngutted f resh Malawian Tilapia ( Oreochromis species ) stored on ice in cooler boxes for 5 d ays Gutt ing DV 1 (w/v)% Day 0 Day 1 Day 3 Day5 Ungutted SEM 0% 7.23 ax 7.20 ax 7.20 ax 7.13 ax 0.03 5% 7.20 ax 7.20 ax 7.20 ax 7.20 ax 0.0 3 8% 7.20 ax 7.20 ax 7.15 ax 7.2 0 ax 0.0 3 10% 7.20 ax 7.15 a 7.15 ax 7.10 ax 0.0 3 SEM 0.0 3 0.0 3 a 0.0 3 0.0 3 Gutted 0% 7.23 ax 7.21 ax 7.20 ax 7.13 ax 0.3 5% 7.20 ax 7.20 ax 7.20 ax 7.15 ax 0.03 8% 7.20 ax 7.20 ax 7.20 ax 7.20 ax 0.0 3 10% 7.20 ax 7.20 ax 7.15 ax 7.15 ax 0.0 3 SEM 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 1 DV = Dried white buffered vinegar a c means in the same column with the di fferent superscript are significantly different (P <0.05) x z means in the same row with different superscript are significantly different (P < 0.05) SEM means standard error of the mean
49 Table 4 6 gutted and gutted Oreochromis species treated with dried buffered vinegar and stored on ice in coolers for 5 days Quality attribute DV 1 (w/v %) Whole ungutted (Days on ice) Whole gutted (Days on ice) 0 3 5 0 3 5 Skin 0 3.67 ax 2.00 cy 1.33 by 4.00 a 2.33 b 2.00 c 5 4.00 ax 2.63 bx 2.33 bx 4.00 a 1.65 c 1.67 bc 8 4.00 ax 3.00 bxy 2.33 ay 3.67 a 3.00 ab 3.00 a 10 3.67 ax 4.00 ax 2.33 ay 3.67 a 3.67 a 2.67 a SEM 0.12 0.12 0.12 0.13 0.14 0.13 Odor 0 4.00 ax 2.00 cy 1.67 by 4.00 a 1.67 b 1.67 b 5 3.67 ax 1.00 dz 2.00 by 4. 00 a 1.67 b 1.00 b 8 4.00 ax 3.00 by 3.30 axy 4.00 a 1.67 b 3.00 a 10 4.00 ax 4.00 ax 2.33 by 4.00 a 2.67 a 2.67 a SEM 0.12 0.13 0.12 0.13 0.14 0.13 Gills 0 3.67 ax 1.67 cy 1.00 cy 4.00 a 3.67 b 1.67 b 5 4.00 ax 2.66 by 2.33 by 3.67 a 2.00 a 1.67 b 8 3.67 ax 3.00 abx 2.00 by 4.00 a 2.33 a 3.00 a 10 4.00 ax 3.67 axy 3.00 ay 4.00 a 3.33 ab 2.67 a SEM 0.12 0.12 0.12 0.12 0.13 0.12 Flesh color 0 3.67 a 2.33 b 3.33 a 3.67 a 1.67 b 1.33 c 5 3.33 a 2.00 b 3.67 a 3.67 a 1.67 b 1.66 bc 8 3.33 a 3.33 a 4.00 a 3.66 a 3.33 a 2.33 ab 10 3.67 a 2.66 ab 4.00 a 4.00 a 2.67 a 3.00 a SEM 0.14 0.14 0.14 0.13 0.14 0.13 Eyes 0 3.33 a 1.67 c 1.00 bc 4.00 a 1.33 b 1.33 b 5 3.67 a 2.33 bc 1.33 b 3.66 a 1.66 b 1.33 b 8 4.00 a 3.00 ab 2.00 ab 4.00 a 2.66 a 1.66 b 10 4.00 a 3.33 a 2.67 a 3.67 a 2.67 a 2.67 a SEM 0.13 0.14 0.13 0.13 0.13 0.13 1 D V = Dri ed white buffered vinegar a,b,c Means in columns within an attribute bearing different superscripts differ significantly (P < 0.05). x,y,z Means in rows within an attribute bearing different super scripts differ significantly (P < 0.05).
50 CHAPTER 5 SUMMARY The objectives of this research were to identify the existing knowledge gaps in fresh fish processing and handling methods among Malawian fish traders by administering a survey (Phase 1), and t o determine the antimicrobial and sensory effects of dried buffered white vinegar powder on storage stability of fresh Malawian Tilapia ( Oreochromis specie s) under ice storage in coolers (Phase 2). The main findings of the study suggest that fish treated with dried white buffered vinegar had lower microbial counts ( aerobic plate count generic E.coli and total coliform count) as compared to the control fish. Increasing levels of dried vinegar from 5% to 10% resulted in decreas ed levels of APC. In terms of APC fish treated with 10 % dried vinegar remained m microbiologically unspoiled as shown by the level of APC at 6.59 log CFU/g on the final day of evaluation (day 5). This was in correlation with sensory observations which indicated that the fish was in goo d quality after 5 days of storage Therefore 10 % dried vinegar may result in extension of shelf life of fresh Tilapia up to 5 days on ice Compared to the control, APC increased from 6 log CFU/g on day 0 to 9 log CFU/g on day 5 .Sensory and quality attribut es for the control were lower than in DV treated samples This trend indicates that the control fish may have been spoiled on day 5 as shown by APC level of 9 log CFU/g and lower quality attributes rating respectively However, further palatability researc h is necessary to demonstrate tha t the DV exten ded the shelf life of the fish. This study demonstrated that g utting of fish prior to DV treatment application resulted in increased levels of APC when compared to un gutted fish This was
51 especially true fo r the 10% DV treatment. In general ungutted fish treated with 5, 8 and 10% DV resulted in 2.65, 2.45 and 3.58 log reductions in APC, respectively when compared to the untreated control (Table 3 1) In contrast, the gutted fish treated with 5, 8 and 10% DV resulted in 2.25, 2.36 and 2.70 log reductions in APC, respectively, when compared to the untreated control. These findings are in agreement to those of a related study by Baixas Nogueras et al ( 2009 ) on the effect of gutting on sensory, microbiological, a nd chemical properties of European hake ( Merluccius merluccius var. mediterraneus ) stored in ice. Gutting resulted in higher counts of Enterobacteriaceae Shewanella putrefaciens and Pseudomonas In terms of chemical spoilage indicators, higher contents o f putrescine, cadaverine, tyramine, and histamine were found in gutted hake. All fish quality indicators studied showed that gutting increased the susceptibilit y of hake muscle to spoilage during ice storage and decreased its shelf life by 4 days. Dried vi negar treatment significantly reduced E.coli counts for gutted and un gutted fish. Increased level of DV resulted in lower numbers of E.coli On day 5 of evaluation, 10 % DV treatment resulted in 1 log r eduction for gutted fish and 2 log reduction for un gu tted fish. Dried vinegar treatment level of 5% did not significanty reduce E. coli contamination when compared to the control (0%). As was the case with aerobic plate count, vinegar treatment significantly reduced (P < 0.05) fecal c oliform counts and there fore reduced fish contamination while gutting did not significantly affect numbers of fecal coliform counts (P > 0.05). In addition to exerting antimicrobial effects on APC, generic E.coli and fecal coliforms, these results suggest that DV also helped to m aintain the quality attributes for gutted and un gutted whole fish. The application of DV did not significantly affect the pH
52 of fish. This study agreed with our hypothesis that the DV treatments would function to reduce microbial growth and improve or mai ntain sensory attributes. C onclusion In conclusion, the 10% application rate of DV to fresh Malawian Tilapia ( Oreochromis species ) can help to reduce microbial spoilage and microbial pathogen contamination and maintain fish quality. This could result in e xtended shelf life and improved fish quality for supply Reduced microbial levels can also lead to enhanced food safety for fresh fish and fish product consumption in Malawi. Future work should be conducted to investigate optimum c oncentratio n of dried vinegar (in excess of 10%) that can be used to improve fresh Malawian Tilapia ( Oreochromis species ) quality and shelf life without affecting organoleptic characteristics. It is important that a sensory evaluation be conducted on the cooked produ ct to determine consumer acceptance in terms of color, flavor, odor and texture, of the fish treated with DV and a shelf life study that monitors the growth of psychrotrophic bacteria (the spoilage microflora) It was also evident from this study that un gutted fish had lower microbial cou nts as compared to gutted fish. This study also revealed the immediate need to provide the Malawian fish processors, traders and merchants, govern m ent and academ ic personnel with access to extension services. The extensio n training should include fresh fish handling, basic hygiene, fresh fish shelf life preservation methods, and knowledge and hands on technology concerning the use of approved food additives to enhance and maintain fresh fish safety and quality
53 Recommenda tion: Considering the present cost of DV, approximated at US $10.0 /Kg, the use of DV or its equivalent could be recommended as a cost effective way of extending the shelf life of fresh tilapia in Malawi. It is also recommended that f uture studies should consider the use of whole ungutted fish. B io g enic amines and other spoilage indices should be used to assess effects of DV .U se of other organic acids, and locally available antimicrobial agents such as lemon juice extract, and garlic extract should be eval uated in future research
54 APPENDIX A SURVEY QUESTIONNAIRE Survey title : Current Methods Employed by Fish Traders in Lilongwe, Limbe and Blantyre City Markets for Fresh Fish Quality Preservation in Coolers and Their Effectiveness in Reducing Fresh Fish S poilage Survey Introduction My name is Matrina Mpeketula; I work with the Fisheries department here in Malawi. I am a graduate student (University of Florida, USA) in the Department of Animal Sciences. As part of my graduate research; I am conducting a st udy on developing methods that will help to delay fish spoilage for fresh fish traders here in Malawi. As part of the study, I am conducting a survey regarding your exp erience in fresh fish spoilage. This survey will be used to better understand the curren t methods being used by you and identify the knowledge gaps in issues related to fresh fish spoilage. You were randomly selected to provide information on among other things the methods that you use to prolong freshness and delay spoilage for fresh fish, t he reasons for using those methods and your general knowledge in food safety and fish handling issues. Please note that your responses will be held with strict confidentiality and no implications will follow you single handedly. Your cooperation is of grea t importance to us as well as to you because you will be briefed on the outcome of this survey. The questionnaire was assigned a number and no reference to the company being interviewed was recorded to maintain anonymity. Immediately following the introduc tion, the participants were asked to give responses to the following thirty questions:
55 A. GENERAL INFORMATION 1. Identification Questionnaire Number: ______ Market : ____________________________________ Name of respondent : ____________________ Phone: ____________ Physical address: __________________________________________________________ Date of interview ___/ ___/ ____ Head of household 1. Yes 2. No If no, what is your relation to the household head? ___________________ 2. Respondent and househol d characteristics 1. Age _______ (Years) 2. Gender 1. Male 2. Female 3. Marital Status 1. Married 2. Single 3. Divorced 4. Widowed 5. Separated 4. Education level ___________ (Years spent in school) 5. Household size ___________ (Number of f amily members) 1. Farmer 2. Civil Servant 3. Employed by company/NGO 4.Other (S pecify) ________________ 7. Main income source 1. Fish business 2. Wage labor 3. Farming 4.Other (S pecify) _______________ 8. Fin ancial capital (value of all business assets) Asset Value Savings Cooler boxes or other (S pecify) Credit (formal or informal)
56 B.FRESH FISH BUSINESS 9. For how long have you been in the fresh fish business? 10. How many t imes per week do you buy fish? 1. Once 2. Twice 3. Three times 4. Other ( S pecify) _________ 11. What are the types of fish species that you prefer selling? 12. What are the reasons for the mentioned species? 13. What type of customers do you mostly sell to? 1. Other traders 2. Consu mers 3. Processors 14. Where do you buy fresh fish? a. Mangochi b. Salima c. Other (Specify) 15 What is the mode of transportation? a. Public transport ( buses, pick up) b. Own vehicle 16. How long does it take to travel from the beach to the city market? 17. How do you av oid fresh fish spoilage? 1. Ice in coolers (If ice is used. refer to 15) 2. Preservatives (S pecify) 3. Gutting 4. Rinsing in clean water 5. Other ( Specify) 6. Nothing 18. If nothing is used, what are the reasons? a. No knowledge of such preservatives
57 b. Expensive c. Consumer prefer ences 19. What do you use for carrying fresh fish during transportation? 1. Cooler boxes 2. Bamboo baskets 3. Plastic pails or Containers 4. Sacks 5. Other ( Specify) 20. How is ice accessed? a. Buy from ice sellers b. Buy from City market ice plant c. Own ice maker 21. How much ice do you use per 1 kilogram of fish? 22. How long do fish take to spoil under your preservation method? Hours Days 23. How do you know that your fish has started spoiling? a. Bad smell b. Sunken eyes c. Gill Color d. Fish scales falling off e. Muscle tenderness / Consistency f. Other ( Specify) 24. What do you do when fish begin to show spoilage signs? 1 Reduce price 2 Sun drying 3 Smoking 4 Other ( Specify)
58 C. Extension services and training 25. Do you have access to extension services? a. (1) Yes (2) No 26. If yes what type of extension service do you access? a. Fish Quality assessment b. Fish Quality preservation c. Fish P rocessing methods 27. Extension services providers a. Government b. c. Others (Specify) 28. How often do you access the extension servic es? a. Once a year b. Twice a year c. More than twice a year d. Never 29. Did you have any formal training for the following? b. Food safety (1) Yes (2) No c. Quality control (1) Yes (2) No d. Fish handling (1) Yes (2) No e. Basic pe rsonal hygiene (1) Yes (2) No 30. What is the main challenge in your fresh fish business?
59 APPENDIX B SURVEY RESULTS SUMMA R Y 11. What are the types of fish species that you prefer selling ? 12. What are the reasons for the preferred species? Great tast e preference high market value cheap producer price Great taste preference and high market value Chambo 83.30% 8.30% 8.30% 0.00% 55.60% 5.60% 5.60% 0.00% Usipa 0.00% 50.00% 50.00% 0.00% 0.00% 5.60% 5.60% 0.00% Chambo, Usipa and Mcheni 33.30% 0.00% 0.00% 66.70% 5.60% 0.00% 0.00% 11.10% Mcheni 0.00% 0.00% 100.00% 0.00%
60 Question 13 to 30 Question Response frequency Percentage 13. What type of customers do you prefer selling to? Consumers 12 66.7 Consumers and other traders 3 16.7 Cons umers and processors 1 5.6 All (consumers, other traders and processors) 3 11.1 14. Where do you buy fresh fish? Mangochi 12 67 Salima 6 33 15.What is the mode of transportation Public transport 18 100 Own vehicle 0 0 16.How long does it take to t ravel from the beach to the city market ( on average) 3 hours 6 33 4 hours 12 67 17.How do you avoid fresh fish spoilage Ice in coolers 11 61
61 Preservatives 1 6 Ice and gutting 5 28 Ice and rinsing in clean water. 1 6 19.What do you use for carry ing fresh fish during transportation Cooler boxes 2 11 Bamboo baskets 11 61 Plastic pails or containers 0 0 Sacks 0 0 All 1 6 20. How is ice accessed? Buy from sellers 18 100 Buy from city market ice plant 0 0 Own ice maker 0 0 21.How much ic e do you use per 1 kilogram of fish 100 grams 1 6 200 grams 3 17 250 grams 2 11 300 grams 2 11 22. How long do fish take to spoil under your preservation method? 1 day 4 22 2 days 3 16
62 3 days 2 11 3 hours 1 6 4 hours 3 17 6 hours 3 17 8 hours 2 11 23. How do you know that your fish has started spoiling? Bad smell 1 6 Sunken eyes 1 6 Gill color 0 0 Fish scales falling off 1 6 Muscle tenderness 1 6 24.What do you do when fish show spoilage signs Reduce price 8 44 Sun drying 1 6 Smoking 1 6 Resale to other processors 3 17 25.Do you have access to extension services and training Yes 1 6 No 17 94 29.Did you have any formal training in the following Food safety 0 0
63 Quality control 0 0 Fish handling 0 0 Basic personal hy giene 0 0 30. What is the main challenge in your fresh fish business? Lack of fish at the lake 3 17 Transportation problems 1 6 Lack of preservation knowledge 6 33 Low selling prices 2 11 Quick spoilage 1 6 Low selling prices 3 17 Quick spoila ge 2 11
64 APPENDIX C SCALE FOR EVALUATING QUALITY ATTRIBUTES O F FRESH MALAWIAN TIL APIA ( O reochromis species ) STORED ON ICE IN C OOLER BOXES FOR 5 DA YS Attribute Highest quality(4) Good quality (3) Fair quality(2) Unacceptable (1) Skin Very intense pigme ntation, transparent mucus Insignificant pigmentation losses, milky mucus Pigmentation without shine, slightly greyish mucus Important pigmentation losses, widely opaque mucus Exterior odor Sharply sea weedy and shell fish Weakly sea weedy and shellfish Incipiently sour and putrid Sour and putrid Gills Brightly red without odor, lamina perfectly separated Rose colored without odor, lamina adhered in groups Slightly dark red, lamina adhered in groups Dark red with bad fish odor, lamina totally adhered F lesh color Red pigmentation in flesh Pinkish pigmentation Pale flesh color No pigmentation Fish eyes Completely clear, glassy, sparkling, turgid Somewhat clear, non glassy, non sparkling Cloudy, traces of bloody spots Unclear, pigmented and fuzzy
65 APP ENDIX D FRESH FISH QUALITY ATTRIBUTES SCORING SCALE FOR FR ESH MALAWIAN TILAPIA Oreochromis species STORED ON ICE IN COO LER BOXES FOR 5 DAYS Instructions: Please rate each sample for fish quality attributes. Circle your response. Sample ID: Sample rating Skin Exterior Odor Gills Flesh Color Fish Eyes 4 (Highest quality) 4 4 4 4 4 3(Good quality) 3 3 3 3 3 2 (Fair quality) 2 2 2 2 2 1 (Unacceptable) 1 1 1 1 1
66 APPENDIX E EXPERIMENTAL LAYOUT FOR DRIED BUFFERED V INEGAR ON APC OF FRE SH MALAWIAN TILAPIA ( Oreochromis species ) STORED ON ICE IN C OOLER BOXES FOR 5 DAYS Treatment DV (%) Days on ice Un gutted whole fish Gutted whole fish 0.0% 0, 1, 3 and 5 Trial 1 Trial 2 Trial 1 Trial 2 Cooler A Cooler A Cooler D Cooler D 5.0% Cooler K Cooler K Cooler G Cooler G 8.0% Cooler W Cooler W Cooler Q Cooler Q 10.0% Cooler M Cooler M Cooler J Cooler J
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72 BIOGRAPHICAL SKETCH Matrina Mpe ketula was born in Dedza, central region of Malawi in 1984. She attended her secondary schoo econdar y S chool in Malawi. She was admitted to the University of Malawi (Bunda College of Agriculture) in 2003 where she graduated with a Bachelor of Science degree in aquaculture and fisheries sciences in 2007. In 2008, she joined the Department of Fisheries under the M inistry of Agriculture. In 2010, she obtained a scholarship under the USAID Initiative for Long Term Training and Ca pacity Building Prog ram to pursue a Master of Science degree in the Department of A nimal S ciences at the U niversity of Florida in U.S.A. She pursued a Master of Science degree in animal sciences focusing on meat processing and food safety under the supervi sion and guidance of Dr. Sally K. Williams