FACULTY OF BIOSCIENCE ENGINEERING

INTERUNIVERSITY PROGRAMME (IUPFOOD) MASTER OF SCIENCE IN FOOD TECHNOLOGY Major Food Science and Technology

Academic year 2014-2015

CHARACTERIZATION OF SALT-FERMENTED FROM THE PHILIPPINES

DULCE FE B. VELASCO

Promoter : Prof. dr. ir. KATLEEN RAES Tutor : ing. ELLEN NEYRINCK

Master's dissertation submitted in partial fulfilment of the requirements for the degree of Master of Science in Food Technology

Copyright

The author and promoters give the permission to consult and copy parts of this work for personal use only. Any other use is under the limitations of copyrights laws, more specifically it is obligatory to specify the source when using results from this thesis.

Gent, 5 July 2015

The promotor The author

Prof. dr. ir. Katleen Raes Dulce Fe Velasco

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Acknowledgement

First and foremost, I wish to express my sincere thanks to my promoter, Professor Katleen Raes, for providing me with all the necessary facilities for the research. Thank you very much for the assistance, understanding, constructive suggestions and corrections, and immense knowledge that greatly improved this paper.

To my tutor, Ellen Neyrinck, my deepest gratitude for valuable guidance and encouragement throughout my entire laboratory works.

I am also indebted to VLIR-UOS for providing financial assistance for my graduate studies here in Belgium.

I take this opportunity to express my appreciation to all the IUPFOOD Coordinators, Prof. Marc Hendrickx, Prof. Koen Dewettinck, Dr. Chantal Smout, ir. Katleen Anthierens, and Katrien Verbist for their help and support.

Many thanks to my Alma Mater & Employer – Mindanao State University, Main Campus especially to the College of Fisheries for all the help during the processing of my application papers and support during the whole duration of my graduate studies.

I am also grateful to a kind-hearted friend, Anna Rose Pilapil, for the encouragement extended to me. Very big thanks for the guidance which helped me in all the time of research and writing of this thesis.

To all my friends who, directly or indirectly, have lent their hand in this venture. Thank you very much for the support and encouragement.

I also thank my parents, spouse and son for the unceasing encouragement, motivation, support and attention that have greatly uplifted my morale especially at those times when I’m jaded.

Most importantly, I am very much grateful to my Lord God, Jesus Christ, and Mama Mary for the good health and wellbeing that were necessary to complete this research paper. Thank You for the unconditional and amazing love that You’ve given to me.

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Abstract

This research comprehensively characterized the salt-fermented or from the Philippines which is an important food product in the country. Anchovy and anchovy paste samples traditionally prepared were obtained in the Philippines and were brought to Belgium for biochemical analyses. Samples were taken from different batches at different time points during fermentation of local producers and samples commercially available in markets. The commercially available anchovy pastes were of 1, 3 and 4-months old. All samples were analyzed for gross composition, pH, water activity, salt content, non-protein nitrogen (free and total NPN), fatty acid composition, mineral content and TBARS for lipid oxidation. The studied bagoong showed a content of DM (38.7±3.52g/100g), protein (50.03±4.86g/100gDM), fat (5.71±1.54g/100gDM), SAFA (45.73±2.31%), MUFA (16±1.33%), and PUFA (28.35±2.63%). The samples have an average pH level of 6.56±0.16. The products were shown to be microbiologically stable with an Aw and NaCl content of 0.82±0.02 and 31.33±6.98g/100gDM respectively. Even if samples were processed in the same country, different results for other parameters like TBARS (11.76±2.43μgMDA/gDM), total NPN (117.89±33.55mg/100gDM), free NPN (21.15±3.15mg/100gDM), and mineral content were observed. The amount and level of proteins, Aw, pH, minerals, PUFA, free and total nitrogen of the raw anchovy decrease at the start of fermentation period while an opposite is observed for DM, fats, salt, SFA and TBARS content. Fermentation increases the concentration of DM, salt content, total and free NPN of the salt-fermented anchovy paste; decreases the content of fats, proteins and TBARS; and no significant effect in Aw, mineral content and fatty acids was observed. Samples taken from different batches at different time points (R, D0, D9, D19 and D28) of a local producer were microbial characterized. The microbial count increased and is at its highest on D19 then decreased gradually on D28. Enterobacteriaceae were not detected. Aerobic bacteria, LAB and halophilic LAB were involved at the start of fermentation whereas proteolytic, halophilic aerobic bacteria and halophilic yeasts played a role starting at the middle fermentation period. The fermentation process was predominated with halophilic bacteria. Halophilic LAB has the highest count. However, no acid fermentation had occurred based on the values of pH obtained.

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Table of Contents

Copyright...... ii Acknowledgement ...... iii Abstract ...... iv List of Abbreviations ...... vii List of Figures ...... viii List of Tables ...... ix Chapter 1. Introduction ...... 1 Chapter 2. Review of Related Literature ...... 3 A. Fermentation ...... 3 B. Fish Fermentation ...... 5 C. Products in Thailand ...... 7 D. Fermented Fish Products in Indonesia ...... 8 E. Fermented Fish Products in ...... 11 F. Fermented Fish Products in Malaysia ...... 11 G. Fermented fish products in Vietnam ...... 14 H. Fermented Fish Products in Philippines ...... 15 I. Fermented in the Philippines ...... 18 Chapter 3. Materials and Methods ...... 20 A. Collected Samples from the Philippines ...... 20 B. Gross Composition...... 21 a. Dry Matter Content ...... 21 b. Crude Protein Content ...... 21 c. Crude Fat Content ...... 23 C. pH ...... 24

D. Water Activity (Aw) ...... 24 E. Mineral Content ...... 25 F. Salt Content ...... 26 G. Fatty Acid Composition ...... 26 a. Anchovy Paste Fatty Acids Extraction ...... 26

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b. Fatty acid Methylation ...... 27 H. Total and Free Non-Protein Nitrogen Content ...... 29 a. Perchloric Acid Extraction ...... 29 b. Total Non-Protein Nitrogen ...... 30 c. Free Non-Protein Nitrogen ...... 30 I. TBARS Analysis ...... 31 J. Microbiological Analysis...... 33 a. Sampling ...... 33 b. Microbial Analysis ...... 33 K. Statistical Analysis...... 34 Chapter 4. Results and Discussion ...... 35 A. Raw materials ...... 35 B. Biochemical and chemical composition of commercially available anchovy pastes ...... 37 a. Chemical Composition ...... 37 b. Mineral Composition ...... 39 c. Fatty Acid Composition ...... 40 d. Non-protein and TBARS composition ...... 45 C. Characterization of fermented anchovy pastes...... 47 a. Chemical Characterization ...... 47 i. Chemical Composition ...... 47 ii. Mineral Composition...... 49 iii. Fatty Acid Composition ...... 50 iv. Non-protein and TBARS composition……………………………………………….53 b. Microbial Characterization ...... 54 Chapter 5. Conclusions and Recommendation ...... 58 Works Cited ...... 60

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List of Abbreviations

ANOVA - analysis of variance Aw - water activity BHI - brain heart infusion Ca - calcium CFU - colony forming units Cu - copper DHA - docosahexaenoic acid DM - dry matter content D0 - day 0 of fermentation period D9 - day 9 of fermentation period D19 - day 19 of fermentation period D28 - day 28 of fermentation period EPA - eicosapentaenoic acid FAME - fatty acid methyl esters Fe - iron FP - anchovy pastes sold in markets/commercially available fish pastes ICP-AES - Inductively Coupled Plasma-Atomic Emission Spectroscopy ISO - International Organization for Standardization K - potassium LAB - lactic acid bacteria MDA - malonaldehyde Mg - magnesium Mn - manganese MRS - de Man, Rogosa and Sharpes’ medium MUFA - monounsaturated fatty acids Na - sodium NaCl - sodium chloride salt NPN - non-protein nitrogen PCA - plate count agar PUFA - polyunsaturated fatty acids R - raw anchovy SFA - saturated fatty acids SP - anchovy pastes with different batches produced from one producer T - timepoints TBARS - thiobarbituric acid reactive substances VRBGA - violet red bile glucose agar Zn - zinc

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List of Figures

Figure 1 Raw material for salt-fermented anchovy paste

Figure 2 Fermented anchovy pastes at different time points

Figure 3 Fermented anchovy pastes locally sold in markets

Figure 4 Microbial count of fermented anchovy pastes on different days of fermentation

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List of Tables

Table 1 Fish Paste and Fish Products in Different Countries (Chinte-Sanchez, 2008)

Table 2 Chemical Composition of some Fermented Fish Products in Indonesia (Putro, 1993; Irianto & Irianto, 1998)

Table 3 Quality Standards of Fermented Fish Products in Myanmar (Tyn, 1993)

Table 4 Composition of Fermented Products in Malaysia (Abdul Karim, 1993)

Table 5 Some Physicochemical Parameters of nuoc-mam (Taira, et al., 2007)

Table 6 Chemical Composition of Bagoong and Patis (per 100g edible portion) (Chinte- Sanchez, 2008)

Table 7 Amino Acid Content of Patis from dilis (mg/100mL) (Chinte-Sanchez, 2008)

Table 8 Chemical Composition of Bagoong dilis (per 100g edible portion) (Chinte- Sanchez, 2008)

Table 9 Characterization of the samples and manufacturers

Table 10 Chemical composition of anchovy paste traditionally produced in some parts of the Philippines

Table 11 Mineral composition of anchovy paste traditionally produced in some parts of the Philippines Table 12 Fatty acid profile (g/100g FAME) of anchovy paste traditionally produced in some parts of the Philippines

Table 13 Fatty acid profiles of anchovy pastes obtained from different studies

Table 14 Non-protein nitrogen and TBARS of anchovy paste traditionally produced in some parts of the Philippines Table 15 Chemical composition of anchovy pastes in different time points of fermentation

Table 16 Mineral composition of anchovy pastes in different time points of fermentation

Table 17 Fatty acid profiles of anchovy pastes in different stages of fermentation

Table 18 Non-protein nitrogen and TBARS of anchovy paste traditionally produced in some parts of the Philippines

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Chapter 1. Introduction

For centuries, food fermentation is a technique used to preserve perishable food products which includes fruits, cereals, vegetables, milk, meat and fish (Hui et al, 2004). Everywhere in the world, about 20-40% of the food supply comes from fermented foods and beverages. Fermentation is a simple and very economical method of food preservation (Chinte-Sanchez, 2008). There are five major fermentation processes: 1) ; 2) acetic acid fermentation; 3) alcoholic fermentation; 4) alkaline fermentation; and 5) adding high amount of salt. According to Steinkraus (1996), fermentation has five roles. These are: a) Human diet enrichment through the development of a wide diversity of food flavors, aromas and textures; b) Food preservation by lactic acid, acetic acid, alcoholic and alkaline fermentation; c) Food enrichment with protein, essential amino acids, essential fatty acids, and vitamins; d) Food detoxification; e) Energy and time reduction.

Fermented fish products are important traditional foods in many countries of the world, particularly in the less developed countries. They are nutritious and available for the consumers at an affordable price. In Asia, fermented small fishes, fish eggs and intestines are widely consumed (Lee, et al, 1993). In the Philippines, fish fermentation is one of the most common methods of due to its simplicity in technique and low equipment cost. Also due to its desirable flavor and cheap source of protein, it has become a part of the diet of most Filipinos. Fermented fish products are fermented through the addition of salt which can be in high amount (15-20%) or in low amount (less than 10%). The former is steered often by fish endogenous enzymes while the latter is steered by lactic acid bacteria. The most popular products of fish fermentation are fish paste and . They have salty, slightly -like flavor and an appetite-stimulating aroma. Fish paste is a naturally fermented whole fish or shrimp with the addition of 20-25% salt under ambient conditions. On the other hand, fish sauce is a straw yellow to amber color liquid extracted through the complete hydrolysis of fish/salt mixture for 9-12 months (Peralta, et al, 2008).

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Fish paste is an indigenous fermented food in the Philippines and locally known as bagoong. It is also called kapi in Thailand, mam in Vietnam, in Burma, padec in Laos, prahoc in Cambodia, jeotkal in Korea, trassi in Indonesia and shiokara in Japan (Chinte- Sanchez, 2008). Any species of fish can be used for the fish paste production. In the Philippines, , which are small fishes and abundant throughout the country, is typically used as a raw material for fish paste. Fermented anchovy paste produces a very distinctive salty and fishy flavor. It is popularly taken not only as side dishes, but also as an ingredient in many types of dressings. It is also used as ingredients in different (Sukuma & Chaiyanan, 2012). However, even though it is popular throughout Asian countries, particularly in Southeast Asia including the Philippines, only limited studies have been conducted regarding fish fermentation unlike other fermentation technologies such as those involving milk and soybeans. Also, as it is traditionally produced, it is artisanal in nature and developed only by trial and error instead of scientific methods. Because of these, they are usually characterized by variation in product quality, efficiency of processes used, and even safety of foods (Lee, et al, 1993). Thus scientific studies are still required for the understanding and improvement, if necessary, of fermented anchovy paste.

This study aims to 1) characterize the biochemical and chemical changes that occur in the traditionally fermented anchovy paste produced in Philippines during the different stages of fermentation; and 2) get insight in the succession of microbial flora involved in the process to better understand the fermentation process.

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Chapter 2. Review of Related Literature

A. Fermentation

Fermentation is derived from the Latin word “fevere” which means “to boil”, as this phenomenon was associated with the production of carbon dioxide bubbles through the anaerobic catabolism of sugars by yeasts when producing alcoholic beverages (Chinte-Sanchez, 2008, Chojnacka, 2010). There are various definitions of fermentation depending on the field of studies. Classical biochemical definition of fermentation is “an anaerobic breakdown of an organic substrate by an enzyme system in which the final hydrogen acceptor is an organic compound”. In a broader sense, it is “a metabolic process in which chemicals are brought about in an organic substrate through the activities of enzymes secreted by microorganisms” (Chinte- Sanchez, 2008). In its simpler definition, as cited by Chinte-Sanchez (2008), it is the processing of foods in which a certain typical desirable characteristic of food develops such as flavor, aroma, and texture as well as to keep its quality by microbial activities. It is also a biotechnological process whereby foods are produced by controlled biochemical reactions from agricultural products. From a biochemistry point of view, it is “an energy-generating process in which organic compounds act as both electron donors and terminal electron acceptors”. Furthermore, for a microbiologist, it is “a process that involves the application of microorganisms to carry out enzyme-catalyzed transformation of organic matter”.

Fermentation is one of the oldest techniques in food preservation. It extends the shelf-life of foods as well as it enhances its flavor and nutritional value (Kilinc et al, 2005). There are a lot of benefits when foods are fermented. Fermentation adds flavor and aroma to foods; preserves the raw material; synthesizes desirable constituents such as vitamins, minerals and other metabolites; increases digestibility, utilization and transportation of essential amino acids thus improving protein quality; changes the physical state of and impart color to the food; reduces antinutritional and toxic compounds such as phytates, tannins, cyanogenic glycosides, saponins, etc.; consumes low energy; uses less capital and operating costs; applies simple technologies; and improves food safety (Chinte-Sanchez, 2008, Chojnacka, 2010, Sahlin, 1999). However, fermentation is sensitive and requires careful control to prevent risk of contamination and

3 intoxication that would result in food safety issues (Chojnacka, 2010). Fermented foods can be eaten cooked or uncooked and even used as (Ishige, 1993).

Most of the ancient fermented foods originated in Central Asia, and then they slowly spread to China, Europe, and other parts of the world. At that time, preservation of foods by fermentation was discovered by chance. There was no explanation how it can preserve natural resources for longer periods. It was during the 19th century that Pasteur discovered fermentation was associated with microbial activities. Moreover, it were the enzymes, produced by microorganisms, that were responsible for the (bio)chemical changes that occur during fermentation. As fermentation changes the physical and chemical characteristics of foods, it does not reduce the quality of foods but rather improves their nutritional value. Thus, these fermented foods have become the only source of nutrients for low-income people particularly in less- developed countries (Chinte-Sanchez, 2008).

Fermentation can be categorized as aerobic or anaerobic based on the oxygen requirement. In an aerobic fermentation, oxygen is required as hydrogen acceptor while anaerobic fermentation doesn’t require oxygen but requires other substances to act as its hydrogen acceptor such as aldehydes or pyruvic acid (Chinte-Sanchez, 2008). In terms of microorganisms used, fermentation can be performed spontaneously, by back-slopping, or by the addition of starter cultures. With spontaneous fermentation, the raw material and its initial treatment encourage the growth of the indigenous flora and a microbial succession takes place. For back-slopping, a new batch of fermentation is inoculated with part of microorganisms used from the previous fermentation batch. This type of fermentation produces a higher initial number of beneficial microorganisms and is faster and more reliable. The addition of a starter culture is often used to inactivate the indigenous flora present in the raw material allowing only the added starter microorganism to grow. Starter cultures can be single, multiple or mixed strain. With single-strain starter, only a single well-defined strain with known technological properties is added. In the case of multiple-strain starter, 2-6 well-defined strains are added while a mixed- strain starter consists of a unknown number of undefined strains (Josephsen & Jespersen, 2004). Microorganisms involved in fermentation are bacteria, yeasts and molds. Bacteria used in industrial fermentations include strains of the following species: Acetobacter, Streptococcus,

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Lactococcus, Leuconostoc, Pediococcus, Lactobacillus, Propionibacterium, Brevibacterium, Bacillus, Microccus, and Staphylococcus. Yeasts include species of Saccharomyces, Candida, Torulopsis, and Hansenula while molds involved are Aspergillus, Penicillium, Rhizopus, Mucor, Monascus and Actinomucor (Chojnacka, 2010).

B. Fish Fermentation

Fresh fish is a highly perishable product due to its biological composition. If it is not immediately utilized or preserved, spoilage will take place. Fish is usually preserved through a combination of different preservation techniques such as smoking, sun-drying, salting, fermentation, grilling and frying (Koffi-Nevry & Koussemon, 2012). Among these methods, fermentation is commonly practiced in Asian countries. Fermented fish products are products of freshwater and marine finfish, , and crustaceans which are processed with salt to undergo fermentation thus, preventing spoilage (Ishige, 1993). Fermented fish differs from as there is a change in the original shape of the fish in the partly liquefied product in the fermentation process (Espejo-Hermes, 1998). Aside from using salt, fish fermentation can be processed with other ingredients, e.g. in combination with , or combined with other methods such as drying (Beddows, 1997; Ishige, 1993; Chinte-Sanchez, 2008). Fermented fish products contribute largely to the protein intake of the world’s population (Beddows, 1997). They are of major importance in Asian countries, like Thailand, Kampuchea, Malaysia, Cambodia, Philippines and Indonesia, which have a bland rice diet. Through the addition of these products in the human’s diet, they serve as a major source of protein (Beddows, 1997; Chinte-Sanchez, 2008).

Fish fermentation is the transformation of organic substances into simpler compounds by the action of either microorganisms or endogenous enzymes (Peralta, et al., 2008; Beddows, 1997), resulting in the production of peptides, amino acids and other nitrogenous compounds. Peptides and amino acids contribute significantly to the typical flavor and aroma of fermented products. These peptides and amino acids were found out to be naturally occurring antioxidants (Peralta et al, 2008).

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Fermented fish products are categorized as lacto-fermented fish; fish sauces; and fish pastes, either ground or unground. Lacto-fermented fish is prepared by fermenting fish with rice or other grains in addition to fish and salt while production of fish paste and fish sauce only involve mixture of salt and fish and is processed together. Fish paste is the fish which is broken down chemically through fermentation characterized as a purée or paste, while fish sauce is the fully hydrolyzed liquid product (Mizutani, et al., 1992). Shrimp, shellfish and crabs can also be used as raw material. Fish paste and fish sauce are unique in South-east Asia. They have been developed as substitute for soybeans which are less easily grown in the region. Moreover, highly salted amino acid or peptide sauces are greatly appreciated in the area due to its umami taste (Ishige, 1993; Chinte-Sanchez, 2008). Protein hydrolysis in fish sauces and fish pastes is due to the fish-gut enzymes instead of proteases from bacteria. Generally, bacterial count naturally present in fish gradually reduces as fermentation goes on in a high salt environment. Halophilic bacteria or osmotolerant yeasts play a limited role in the development of flavor or aroma (Lee et al, 1993). Fish pastes and fish sauces vary from different countries in raw materials, flavor, and physical properties (Table 1) (Chinte-Sanchez, 2008).

Table 1. Fish Paste and Fish Sauce Products in Different Countries Country Fish/ Fish/Shrimp Sauce China Yu-lu

Cambodia Prahoc Nuoc-mam Indonesia Trassi; Trassi-ikan; trassi udang Ketjap-kan; Kecap-ikan Japan Shiokara Korea Jeotkal Jeot-kuk Laos Pradec Padec Malaysia Belachan ; Sambal-ikan Myanmar (Burma) Ngapi Mga ngan-pya-ye; Hymin ngan-pya-ye Philippines Bagoong/Alamang Patis Thailand Kapi Nampla Vietnam Mam-ca; Mam-ton mam Nuoc-mam Source : (Chinte-Sanchez, 2008)

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C. Fermented Fish Products in Thailand

Thailand is a seasonal tropical climate country which is very dry with only 3-4 months of rainy season thus, food availability for the whole year is a problem. Because of this, food storage and preservation become important factors for maintaining food supplies. Fermentation is one of the techniques practiced by Thai people in preserving their food. These fermented foods play an important role in Thai diet. Their fermented fish products include nam-pla (fish sauce), budu (cloudy fish sauce), kapi (shrimp paste) and plaa-raa (salt-fermented fish) which vary greatly from community to community and between regions handed from generation to generation. These foods are usually produced for family consumption but nowadays, it has moved to small factory production. Fish fermented products in Thailand are classified into three groups: a) fish with a large amount of salt; b) fish with salt and carbohydrate added; and c) fish with salt and fruit added. Popular products under the first group are nam-pla, kapi and budu. Plaa-som, plaa-raa, plaa-chao and plaa-paeng-daeng are some products of the second group while khem-bak-nad and plaa-mum are examples of the third group (Phithakpol, 1993).

For the first group, marine fishes are mostly used. Nam-pla could also be made from and freshwater fish but the best quality is made from anchovies. The ratio of salt to fish/shrimp is 1:3 for nam-pla and budu, while 1:3-5 for kapi. The microorganisms responsible for fermentation of nam-pla were believed to be Staphylococcus, Bacillus and Sarcina sp.; and Pediococcus halophilus, S. aureus and S. epidermidis for kapi and budu (Phithakpol, 1993). As cited by Chinte-Sanchez (2008), an initial count of 104 CFU/mL of aerobic bacteria has been observed in nam-pla. After 3 weeks of fermentation, total bacterial count increases up to 108 CFU/mL then decreases to a non-detectable level after 6 months. She also cited that Bacillus, Micrococcus, Staphylococcus, and Halobacterium sp. were isolated in nam-pla. The latter is involved in the maturation of nam-pla. On the other hand, Thienchai & Chaiyanan (2012) have identified Pediococcus acidilactici, Tetragenococcus halophilus, Lactobacillus plantarum and Lactobacillus delbrueckii from kapi.

In the second group, freshwater fishes are mainly used but freshwater shrimp could also be used. Cooked, roasted or fermented rice are added as source of carbohydrate for the

7 microorganisms involved in the fermentation, such as lactic acid bacteria, and for taste development of the product. Products with added include plaa-som, koong-som and som-fak. The cooked rice can be added whole or minced to fish with salt. Microorganisms involved are Lactobacillus plantarum, Saccharomyces and Candida sp. Plaa-raa, plaa-chom and koong-chom uses coarse-ground roasted rice, from glutinous or normal type of rice, added to fish after few days of salt fermentation. Roasted rice provides browning and specific flavor of the final product. Pediococcus halophilus, Staphylococcus epidermidis, Micrococcus and Bacillus sp. were involved in the fermentation process. Plaa-chao and plaa-paeng-daeng are added with fermented (khaou mak) and ang-kak rice (red rice) to fish respectively. Khaou mak is prepared by mixing the rice with yeast balls while ang-kak is by rice mixed with mold, Monascus purpureus, giving the distinct red color and flavor. Microorganisms involved in the early stage of fermentation of plaa-chao are Bacillus, Staphylococcus, Saccharomyces and Endomycopsis sp. while Pediococcus cerevisiae is involved in the later stage. For plaa-paeng- daeng, Lactobacillus, Micrococcus and Saccharomyces sp. were involved. Under the third group, khem-bak-nad/khem-mak-nad is a salted fish (1:1-5 fish:rock salt ratio) which were cut into long, thin pieces packed tightly in a container and left overnight. On the next day, chopped pineapple is added and placed in bottles and then fermented for three months. On the other hand, plaa- mum is prepared by mixing salt to small-cut pieces of fish (1:3 salt:fish) then mixed with ground roasted rice, tightly packed in containers for 1.5-2 months after which, fish is repacked in containers added with chopped papaya and ground galangal (Phithakpol, 1993).

D. Fermented Fish Products in Indonesia

Fermented fish products in Indonesia have gained special popularity in the local markets. Among the popular fermented fish products in Indonesia are trasi (shrimp or fish paste), pedah (fatty, partly dried, salty fish), kecap ikan (fish sauce), jambal roti (fermented dry salted marine ) and bekasam (fermented freshwater fish) (Table 2) (Putro, 1993).

Pedah has a reddish brown color, slightly wet and pasty with a flabby texture, and a cheesy, salty flavor often mixed with a mild rancid flavor. It is made from salting (1:3 salt:fish),

8 drying and fermenting fish, usually from mackerel. It requires salted fish to be wet, stacked in baskets or other suitable containers for slow dehydration leading to maturation and development of desirable flavor and texture for about 2-3 months at 29°C. However, the fermentation period of pedah varies depending on the processor. As cited by Putro (1993), internal organs of fish stimulate protein and lipid degradation. However, evisceration will facilitate oxidative rancidity due to a larger surface area. With regards to fatty acids, there were more PUFA losses, particularly C22:6n-3 and C22:5n-3, during salting while C20:5n-3 was just stable. Most monounsaturated and saturated fatty acids also decreased except C18:1 and C22:1. Microbial analysis showed that gram positive cocci predominate in the fermentation process followed by lactic acid bacteria. He also mentioned that anaerobic condition speeds up the reddish-browning of the product due to Malliard reaction, hinders oxidative rancidity, facilitate protein breakdown, and microbial population of pedah.

Trasi is prepared from planktonous shrimp Schizopodes or Mytis sp. and small fishes such as Stelophorus or Engraulis sp. Trasi udang (shrimp paste) are more popular than trasi ikan (fish paste). It is used as an appetizer and also eaten with chili, and salt called sambal. Trasi udang production starts at the time the shrimps are harvested and placed on the fishing vessels where 10% salt is added, then another 5% salt is added upon arrival on the landing area. Shrimps are then partially dried under the sun on mats for 1-3 days. When moisture content has decreased to about 50%, shrimps are kneaded and mixed, then sun-dried and kneaded again. At the same time, a coloring agent, like carthamine D or rhodamine B, is often added. Using cylindrical-formed nipa leaves, the paste is pressed and then fermented until the desired trasi aroma is achieved. However in other places like Java Island, shrimp paste is processed differently. Raw or pre-cooked shrimps are added with 15% salt and partially sun-dried for only 1 day then minced, mixed and kneaded forming a paste called brabon. This brabon is sun-dried and kneaded again until it achieves a fine thick homogenous paste which is then pressed in cylindrical-formed bamboo and fermented until the desired aroma is attained. Trasi ikan is prepared with the same process as trasi udang but the former is more often added with coloring agents and has a stronger smell, making it less popular to the locals than the latter. In a study of trasi powder, the total bacterial count reduces during the 7-day fermentation while lactic acid bacteria are just constant. In addition, Staphylococcus, Bacillus and Proteus were also present in

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the product. It was pointed out that changes in total volatile base (TVB) and pH of trasi and trasi powder was not significant. Yet, trasi powder is less accepted by consumers than the normal shrimp paste (Putro, 1993).

Kecap ikan is made from oil sardines (Sardinella sp.) where 25-30% salt is added and then fermented. After fermentation, it is filtered to separate the solid and liquid part. This liquid part comprises the fish sauce, which is mixed with brown sugar and spices. However, fish sauce has become less popular in the country due to competition with . Bekasam is made from freshwater fishes. Its production involves evisceration, splitting of fish into butterfly shape, brining (± 15% w/w) for 3 days, draining, mixing with roasted rice, packing, and then fermentation. However, the process differs from place to place. Jambal roti is prepared from marine catfish (Arius sp.) involving salting (pickling), splitting of the pickled fish, drying on interwoven bamboo mats and fermentation. During the second or third day of drying, fishes are split again and then turned occasionally. However, the production varies slightly from region to region. Moisture and salt content of jambal roti varies between 40-50% and 20-25% respectively. Fermented fish products are household-based processed, thus, the quality of even same products differs from place to place (Putro, 1993). There has been a need for further studies for a standardization of the quality and safety fermented fish products.

Table 2. Chemical Composition of some Fermented Fish Products in Indonesia Shrimp Fish Components Pedaha Bekasamd Jambal rotie Pasteb Saucec Moisture (g/100g sample) 53.83 3,0-5 66-76 52-66 49.27-49.68 Crude Fat (g/100g sample) 9.63 2-5 0.5-0.7 1-23 0.69-1.19 Crude Protein (g/100g 52.12 20-40 10-10.5 41-64 54.17-61.86 sample) NaCl (g/100g sample) 19.21 23 25-30 6-17 7.38-8.53 Total Ash (g/100g sample) nd 10-40 21-23 13-28 34.93-38.80 Carbohydrate (g/100g sample) nd 3.5-5 0.3-1.5 nd nd pH 6.5 nd nd 4.46-4.98 6.57-6.91 Sources : a,b,c As cited by Putro (1993) : aSyachri and Anwar , 1977 ; b Moeljohardjo, 1972; c Poernomo et al., 1984; d Putro, 1993; eIrianto & Irianto, 1998; nd = not determined

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E. Fermented Fish Products in Myanmar

Fish fermentation is a centuries-old food preservation technique in Myanmar and the product is commonly known as nga-pi which has become their national food. Nga-pi is prepared from fish or shrimp, pounded or ground, added with salt and partially sun-dried for 3-4 days. The mixture is pressed and stored in earthen jars or concrete vats for 3-6 months of maturation. The liquid formed during fermentation is also utilized and called as ngan-pya-ye (fish sauce). It is kept in tanks for 3 months to 1 year for aging. After aging, it is boiled for 4-6 hours for partial sterilization of the sauce and reduction of moisture content up to 55-60%. As cited by Tyn (1993), nga-pi and ngan-pya-ye are rich in essential amino acids. Due to the fact that fermented fish has long been existed in Myanmar, and even labeled as their national food, standards were formulated for the quality of the different products. Table 3 shows the different specifications of the various fermented fish in Myanmar. Microbiological analysis of hmyin nga-pi and hmyin ngan-pya-ye reveal that Arachnia propionica, Bacillus sp., Micrococcus sp., and Nocardia dentocariosa were present in both products. In addition, Staphylococcus epidermidis and Corynebacterium sp. were also present in hmyin nga-pi (Tyn, 1993).

Table 3. Quality Standards of Fermented Fish Products in Myanmar

Component Fish Paste Fish Sauce Shrimp Paste Shrimp Sauce Moisture (g/100g sample) 40 55 40 55 Crude Fat (g/100g sample) 1.5 1 1.5 1 Crude Protein (g/100g sample) 18 18 18 18 NaCl (g/100g sample) 25 25 25 25 Source: Tyn, 1993

F. Fermented Fish Products in Malaysia

Post-harvest losses is one of the common and critical problems faced by Malaysian fishing industry. Through food preservation, such as fermentation, these losses will be significantly reduced. However, traditional techniques are still being practiced in Malaysian fish

11 fermentation industries which were managed usually by family members of small entrepreneurs resulting in poor hygiene and quality products. Thus, further studies are required to enhance the fish fermentation industry of the said country (Abdul Karim, 1993).

Fermented products in Malaysia include budu (fish sauce), kicap ikan, pekasam, belacan (shrimp paste), and cincaluk. Budu is a dark brown liquid extracted from salt-fermented fish, particularly from small anchovies (Anchoviella commerson and A. indica), which is rich in salt and soluble nitrogen compounds with a distinctive odor and flavor. It is mainly used as flavoring agent and . It is prepared by washing first the anchovies in seawater and then mixed with salt (1:2-3 salt:fish). The mixture is left to ferment anaerobically in earthenware containers or concrete vats for 3-12 months at ambient temperature with occasional stirring. During this period, fish proteolysis is happening along with the development of the typical budu aroma. At the end of the fermentation period, the formed liquid supernatant is mixed with the fish residues and boiled in addition of coconut palm sugar, , and other flavoring ingredients. Palm sugar eliminates the fishy smell and improves odor and taste of budu, while tamarind reduces the pH which inhibits putrefactive bacterial growth aside from enhancing the flavor of the product. After cooling the boiled fish sauce, it is filtered and bottled. Kicap ikan is manufactured in the same way as budu except that it is made from other types of fish such as goatfish (Upeneus sp.) and herring (Clupea sp.). Stainless steel containers with tight-fitting lids were used instead of concrete vats or earthenware containers (Abdul Karim, 1993).

Pekasam is made from fermenting freshwater fish with roasted rice, tamarind and salt which is usually consumed deep-fat fried or as a side-dish. Marine fish could also be used as raw material. It is prepared by cleaning the fish, mixing with salt (20-50%) then left overnight, mixing with roasted rice (50% w/w) and some tamarind after draining, and packed tightly in earthenware or similar containers for fermentation of 2-4 weeks. Roasted rice aids in masking the fishy odor, promotes development of the characteristic color of pekasam, and serves as the source of carbohydrates for the growth of Lactobacilli. During fermentation, there’s an outgrowth of lactic acid bacteria which lowers the pH, thus, together with the presence of salt, preserving the product. Organic acids produced, particularly lactic acid, also aid in the flavor development of pekasam. Fish protein is also broken down into peptides and amines during the

12 fermentation process, where together with acids and other microbial fermentation products, leads to the development of the typical odor and flavor of pekasam (Abdul Karim, 1993).

Belacan is made from small shrimps (Acetes and Mysid sp.) which is prepared by mixing shrimps with salt (10-15% w/w), sun-dried (5-8 hours) on mats until 50% moisture content is reached, minced or pounded into blocks or paste in wooden tub or a similar container, then fermented for 7 days. Thereafter, the paste is broken down again to small pieces and sun-dried further for 5-8 hours followed by second mincing, packing tightly into balls or other desired shape, and then fermented again for a month. These drying, mincing and fermenting processes can be repeated many times when needed. The finished product is grounded and packed into desired sizes and shape which usually has a dark color, salty taste and strong shrimp odor. Abdul Karim (1993) cited that the bacteria involved in belacan fermentation were Bacillus, Pediococcus, Lactobacillus, Micrococcus, Sarcina, Clostridium, Brevibacterium, Flavobacterium and Corynebacterium where the predominant bacteria were lactic acid bacteria, Micrococcus, Bacillus and high salt tolerant species.

Cincaluk is a fermentation product of small shrimps (Acetes sp.) with salt and cooked rice. It has a pale pinkish color, strong characteristic flavor and salty taste. It is manufactured by washing the shrimps first in seawater, draining and mixing with salt (20-25%) and cooked rice (6% w/w weight), packing in covered earthenware or suitable containers, and then a fermentation of 20-30 days until pink-colored shrimp is achieved. The locals usually use cincaluk as dips, sauce, and flavoring ingredient, and consumed it with rice (Abdul Karim, 1993).

Table 4. Composition of Fermented Products in Malaysia Components Budua Pekasamb Belacanc Moisture (g/100g sample) 54.8-76 57.0-73.0 27.0-40.0 Crude Fat (g/100g sample) 0.2-1 3.0-8.0 1.4-2.6 Crude Protein (g/100g sample) 5.8-11.5 15.0-25.0 28.7-40.0 NaCl (g/100g sample) 21.7-28.15 10.0-16.0 13.0-25.3 Ash (g/100g sample) 18.3-20.9 6.0-14.0 20.0-27.6* pH 5.4-6.2 4.5-6.1 7.2-7.8 Source: aChia Joo Suan (1977) ; bZaiton (1980); cMerican et al. (1980); a,b,c cited by Abdul Karim, 1993; *including salt

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G. Fermented fish products in Vietnam

The most important fermented fish product in Vietnam is nuoc-mam, a fish sauce It is consumed largely in the country and mostly added to rice. It has a clear brown color, salty taste and a distinctive meaty aroma (Beddows, 1998). It is prepared from small fishes, mainly from clupeids and carangids like Decapterus, Engraulis, Dorosoma, Clupeodes, and Stolephorus sp., mixed with high amounts of salt and fermented in earthenware containers for several months (Prajapati & Nair, 2003, Van Veen, 2012). Amino acids are responsible for the development of the characteristic flavor of nuoc-mam. The taste-active components of nuoc-mam, as identified by Park, et al. (2002) were glutamic and aspartic acid, threonine, alanine, valine, histidine, proline, tyrosine, cystine, methionine, and pyroglumatic acid. Many of these components were responsible for the umami, sweet, and overall taste of nuoc-mam. Glutamic acid contributes most of the flavor followed by pyroglutamic acid and alanine. Table 5 shows some physicochemical characteristics of nuoc-mam. Uchida, et al. (2004) have isolated Bacillus subtilis from Vietnamese fish sauce while Bacillus vietnamensis sp. nov. were isolated by Noguchi, et al. (2004).

Another fermented fish product in Vietnam is mam which is a nitrogen-rich fish paste. Its preparation is comparable with bagoong of the Philippines except that after the removal of the liquid (nuoc-mam), the fermented fish is coated with rice flour (thinh) and a film of sugar (chao mam) and again undergo fermentation (Chinte-Sanchez, 2008).

Table 5. Some Physicochemical Parameters of nuoc-mam NaCl (g/100mL) 26.1 pH 5.3 Total Nitrogen (g/100mL) 2.3 Volatile Basic Nitrogen (mg/100mL) 421 Source : Taira, et al., 2007

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H. Fermented Fish Products in Philippines

As an archipelago, the Philippines is rich in aquatic resources, particularly fish and shellfish. Just like any other Asian country, fish fermentation is popular in the Philippines for preservation of excess of foods brought by seasonality of the fish, and lengthening the shelf-life of foods. The most common fish fermented products in the Philippines are fish paste (bagoong), shrimp paste (bagoong alamang), fish sauce (patis), fermented fish-rice mixture (), and fermented shrimp-rice mixture (balao-balao). Bagoong, bagoong alamang and patis are produced at large scale and even exported to other countries while burong isda and balao-balao are just popular in some parts of the country (Mabesa & Babaan, 1993).

As cited by Chinte-Sanchez (2008), bagoong and patis are manufactured in the same way although the latter has a longer fermentation period to which fish flesh is allowed to further disintegrate until it reaches a liquid form. However, in many cases, bagoong and patis are processed together where the former constitutes the solid part while the latter is the liquid part (Chinte-Sanchez, 2008). Both products are made from anchovies (Stolephorus commersonni, S. indicus), sardines (Sardinella fimbriatan, S. longicep), roundscad (Dacapterus macrosoma), herring (Clupeiodes lila), and mackerel (Rastrilliger neglectus). In the Philippine standard (1984), bagoong is defined as “a mixture of salt and small fish or small shrimp, with or without added condiments and flavoring or coloring agents, which have undergone partial or complete fermentation”. Bagoong na isda is termed when it is prepared from fish, bagoong alamang from shrimp, and bagoong na sisi from shellfish. Bagoong na isda has a dark grey color, pasty consistency, and cheesy flavor with traces of fishy odor. Bagoong alamang and bagoong sisi also have the same characteristics as bagoong na isda. Though the initial color of shrimp is pink, it turns grayish when fermented. Thus, red food color is added to make bagoong alamang appealing to consumers. Bagoong, unlike patis, is only hydrolyzed partially making the shape of the raw material still distinguishable. Bagoong and patis are prepared by mixing salt to fish (1:2- 3 or 2:7 salt:fish) then putting the mixture in vats or concrete tanks, allowing them to ferment for 30-90 days in the case of bagoong, and 6-12 months for patis at 28-32°C. Extraction of proteins from the fish is influenced by the pH of the fermentation mixture where a maximum extraction is obtained at pH 7-9, with a very rapid decrease from pH 6 to pH 5. Protein will also

15 precipitate when salt concentration is 20% or more. Microbial analysis of fermented fish paste and fish sauce, cited by Chinte-Sanchez (2008), shows that non-salt-tolerant and salt-tolerant microorganisms are present with halophilic bacteria playing the key role in the fermentation process. Aerobic gram-positive and gram-negative microorganisms dominate in the initial stage of fermentation. Bacillus sp. predominates throughout the fermentation period. In patis fermentation, Bacillus coagulans, B. megaterium and B. subtilis predominate at the early stage while Bacillus licheniformis, Micrococcus sp., Staphylococcus epidermidis and S. saprophyticus are found at the later stage of fermentation. The total viable counts decrease rapidly up to the sixth month of fermentation and slow decline towards the end. The chemical composition of bagoong and patis is shown in Table 6. Chinte-Sanchez (2008) have cited the changes in pH, salt concentration, total nitrogen, formaldehyde nitrogen, ammonia nitrogen, amino nitrogen, and acidity of patis made from Stolephorus sp. mixed with Sardinella and Rastrilliger sp. fermented for 1 year. The pH ranges from 5.97 to 6.5 and acidity is between 0.67-1.42% which shows no interdependency between acidity and pH. Salt content is between 26-27% for 3-12 months of fermentation. Total nitrogen, formol nitrogen, ammonia nitrogen, and amino nitrogen decreases with fermentation time. There has been an increase in the amino acid content of patis made from Stolephorus sp. from the first to sixth month of fermentation, with a slight decrease on the ninth month, and then increases at its peak at the end of fermentation. Table 7 shows the amino acid content of patis from Stolephorus sp. and mixed fish species at various stages and revealed that cystine and proline were absent. Cystine is absent possibly because of oxidation or bacterial action, while proline could have been metabolized during spoilage (Chinte-Sanchez, 2008).

Burong isda is made from mixing rice, boiled dry or cooked to a porridge-like consistency), fish (freshwater fish, , Ophicephalus striatus, mossambica, Therapon plumbeus), and salt, with or without angkak (red rice), fermented for days or weeks depending on the salt concentration. Balao-balao is made in the same way as burong isda except that fermented shrimp of species Macrobrachium or Peneaus is mixed with rice and salt rather than fish (Mabesa & Babaan, 1993).

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Table 6. Chemical Composition of Bagoong and Patis (per 100g edible portion) Component Bagoong ginamos Bagoong balatohan Bagoong padas patis Moisture (g) 55.4 30 65.9 66.3 Fat (g) 1.8 3.2 1.7 0.3 Protein (g) 23.4 8.1 9.6 10.6 Ash (g) 19.4 32.8 22.8 21.9 Carbohydrate (g) 0 25.1 0 0.9 Calcium (mg) 821 1770 504 42 Phosphorus (mg) 510 439 435 32 Iron (mg) 8.2 11.9 16.6 9.3 Source : Chinte-Sanchez, 2008

Table 7. Amino Acid Content of Patis from dilis (mg/100mL) Composition 6 months 12 months threonine 306 612 isoleucine 404 600 leucine 505 601 lysine 554 1083 methionine 204 339 cystine 0 0 phenylalanine 197 365 tyrosine 31 54 valine 355 770 arginine 102 431 histidine 352 976 alanine 318 625 aspartic 586 960 glutamic 586 960 glycine 165 522 proline 0 0 serine 188 431 Source: Sanchez and Klitsaneephaiboon 1983 cited by Chinte-Sanchez 2008

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I. Fermented Fish Paste in the Philippines

Fermented fish paste is locally known as bagoong in the country. The final product varies from region to region. In Tagalog provinces, bagoong is completely fermented, ground, with or without addition of coloring agent. In Ilocano and Pangasinan provinces, bagoong is partially or completely fermented with the entire fish still intact. In Visayas or Mindanao, bagoong is just slightly fermented and without liquid, fishes are still hard and firm and salt is still visible. Among the types of fishes mentioned above as raw materials for bagoong making, anchovies (locally called as dilis) are the most preferred one because it results to a product with pleasing aroma and taste. As discussed earlier, bagoong is manufactured by mixing dry salt with fish (1:2- 3 or 2:7 salt:fish). The fish is cleaned and washed first then dried before adding the salt. The salt- fish mixture is then placed to vats or tanks for fermentation for 30-90 days at 28-32°C (Chinte- Sanchez, 2008). However, the process is done traditionally and varies between processors. Some processors doesn’t wash the fish anymore, especially those freshly harvested fish, and drain the salt-fish mixture before placing into large plastic drums rather than concrete tanks.

Bagoong is primarily used as a condiment and in some places as a staple food. It contains 8-25% protein making it a good source of protein (Mojica, et al., 2005). Mojica, et al. (2005) also studied the effect of irradiation in production of fermented fish paste from dilis. Their results showed that non-irradiated fermented fish paste attains a total plate count of 1.2x102 cfu/g while 3.6x10-1cfu/g and 9x10-1cfu/g were obtained from 3kGy and 10kGy irradiated fish paste respectively. Table 8 shows the different chemical composition of bagoong dilis.

Microbial interaction in bagoong fermentation involves gram-negative rods as initial flora of fish-salt mixture from fish itself and handlers which were inhibited immediately upon the addition of salt due to water extraction by osmosis. The halophilic bacteria, in viscera and gills of fish and those introduced with salt, increase rapidly due to nutrient availability in the brine. Protein hydrolysis and production of flavors are caused by enzymatic action (endogenous) produced from Bacillus subtilis and B. coagulans. The following micoorganisms, and the enzymes they produced, are responsible for fat oxidation leading to the formation of volatile fatty acids : Bacillus licheniformis, Micrococcus colpogenes, and Staphylococcus epidermidis

18 found at the middle stage of fermentation; Micrococcus roseus, M. varians, and Staphylococcus saprophyticus at the later stage; and Bacillus pumilus dominating throughout the fermentation process (Chinte-Sanchez, 2008). It was also cited by Banaay, et al., (2013) that the lactic acid bacteria, Pediococcus halophilus, was involved in bagoong fermentation.

Table 8. Chemical Composition of Bagoong dilis (per 100g edible portion)

Moisture (g) 67.1 Protein (g) 10.3 Fat (g) 1.9 Ash (g) 20.7 Calcium (mg) 535 Phosphorus (mg) 313 Iron (mg) 10.9 Source: Chinte-Sanchez, 2008

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Chapter 3. Materials and Methods

A. Collected Samples from the Philippines

Raw anchovies and salt-fermented anchovy pastes weighing 250 grams each, and liquid extracts at different time points (0, 9, 19 and 28 days) were obtained from a local producer in the province of Misamis Oriental, Philippines. Three different batches were sampled. Also three finished products locally sold in the market were purchased from the province of Surigao, Philippines.

Anchovies that were used for the preparation of the different salt-fermented fish belonged to the Stolephorus species (figure 1). Traditional way of fermentation is done by placing the anchovies in a fine mesh strainer to drain the excess of sea water and to remove visible debris. Still in the strainer, anchovies are mixed with salt in a ratio of 1:3-4 (salt:fish) until the fish becomes not slimy anymore and left for 30-45 minutes to drain the liquids leached from the fish. The mixture is then transferred to clean plastic drums covered first with plastic cellophane, then followed by the cover of the drum. It is then stored at a closed room with a temperature of 28- 31°C for fermentation which takes place for a period of weeks to months. Products can already be sold to consumers after one week, but mostly, they are sold after at least one month of fermentation.

Sampling was done from July 16th to August 13th, 2013. All collected samples were stored under low temperature conditions (-18°C) and preserved at ± 4°C for transportation to Belgium where they were stored at -20°C until analyses. Raw anchovies and salt-fermented anchovy pastes were homogenized first before being analyzed.

Figure 1. Raw material for salt-fermented anchovy paste

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B. Gross Composition a. Dry Matter Content (ISO 1442-1973)

Material: Drying oven at 103±2°C Analytical balance Desiccator Preheated sea sand Aluminum foil cups Raw anchovies samples or salt-fermented anchovy pastes

Reagents: 99% ethanol

Procedure: To aluminum foil cups, 15 grams of sea sand was added. The cups were preheated in the oven at 103°C for one hour, then they were cooled down in a desiccator for at least 45 minutes and weighed (=M0). To the cups, 5 grams of raw anchovies or salt-fermented anchovy pastes were added and weighed again (=M1). Samples were then mixed with 5mL of ethanol and placed in the oven for 3 hours after which they were cooled down in a desiccator for at least 45minutes and weighed (=M2). Dry matter content was calculated as:

(푀 − 푀 ) 퐷푀 = 2 0 푥 100 (푀1 − 푀0)

Where: DM = dry matter content (g/100 gram sample) M0 = mass of the preheated sea sand in the aluminum cup (g) M1 = mass of sand and sample in the aluminum cup before drying (g) M2 = mass of sand and sample in the aluminum cup after drying (g)

b. Crude Protein Content by Kjeldahl Method (ISO 937-1978)

Material: Analytical balance Nitrogen-free paper Destruction tubes Digestion system (Büchi) Distillation unit (Büchi) Burette

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Conical flasks (250 mL) Raw anchovies samples or salt-fermented anchovy paste

Reagents: Kjeldahl tablets (consisting of 235g Na2SO4, 4g CuSO4.5H2O and 5g selenium powder) 98% Concentrated sulfuric acid (H2SO4) Tashiro color indicator (1g methyl red and 0.5g methylene blue dissolved in 500mL ethanol) Phenolphthalein color indicator 32% Concentrated sodium hydroxide (NaOH) 0.16M Boric acid (10g H3BO3 added with 0.4L distilled water and 0.2L ethanol for 1 liter solution) 0.1M Hydrochloric acid (HCl)

Procedure: On a nitrogen-free paper, 1 gram of sample was weighed and placed in a destruction tube, followed by adding a Kjeldahl tablet and 20mL of H2SO4. The mixture was heated in the destruction chamber for 1 hour until a clear green solution was obtained. Destruction tubes were cooled down, after which 50mL distilled water and 4 drops of phenolphthalein indicator were added. After cleaning the distillation unit with distilled water, the tubes were attached. To each tube, (32%) NaOH was supplied until the solution turned dark red to brown. A conical flask with 50mL boric acid and 4 drops of tashiro indicator was placed in the outlet of the distillation in which the distillate was collected. Each sample was distilled for exactly 4 minutes. The purple boric acid solution turned from purple to gray to green color. The flask with the distillate was then titrated with 0.1M HCl until the color turned back to purple. The amount of HCl titrated was recorded. Crude protein content was calculated as:

(푉 − 푉푏) ∗ 14 ∗ 푁 ∗ 6.25 퐸 = ( ) ∗ 100 푉푠 ∗ 퐷푀 ∗ 1000

Where: E = crude protein content (g/100gDM) V = volume of HCl titrated for the sample (mL) Vb = volume of HCl titrated for the blank (mL) Vs = weight of the sample (g fresh sample) N = normality of HCl (mol/L) 14 = molecular weight of nitrogen (g/mol) 6.25 = protein conversion factor DM = dry matter content of sample (g/100g fresh sample)

22 c. Crude Fat Content by Soxhlet Method (ISO 1444-1973)

Material: Extraction thimbles Aluminum foil cup with dried sample (obtained by dry matter determination ISO 1442-1973) Cotton wool Soxhlet apparatus Extraction flasks Analytical balance Drying oven 103±2°C Desiccator

Reagents: Petroleum ether (bp 40-60°C)

Procedure: The extraction flasks were first dried at 105°C for 1 hour and were cooled down afterwards in a desiccator for at least 45 minutes and then weighed (=K1). The aluminum foil cups with dried samples, obtained by dry matter analysis (ISO 1442-1973), were placed in the extraction thimbles and covered with cotton wool. In the Soxhlet apparatus, the thimble was placed in the thimble holder to which petroleum ether was added in such a way that the extraction flask contained two times the volume of the thimble holder. The apparatus was heated for 6 hours with petroleum ether. Normally all the fat was extracted after 6 hours as seen at the bottom of the extraction flask. Petroleum ether was collected and removed until all the extracted fat remained in a minimum amount of petroleum ether. Extraction flasks were then dried at 103°C for 1 hour after which they were cooled down in a desiccator and weighed (=K2). Crude fat content was calculated using the formula:

퐾 − 퐾 푉 = [( 2 1 )⁄퐷푀] 푥 100 푀1 − 푀0 Where: V = fat content (g/100gDM) K1 = weight of the empty flask (g) K2 = weight of the flask with extracted fat (g) M0 = mass of the preheated sea sand (g) from the dry matter analysis M1 = mass of sand and sample before drying (g) from the dry matter analysis DM = dry matter content of sample (g/100g fresh sample)

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C. pH (Bendall, 1978)

Material: Ultra Turrax pH meter (Consort C830) Plastic sample cups Raw anchovies samples or salt-fermented anchovy pastes

Reagents: Bendall solution [9.3mg iodoacetic acid (C2H3IO2), 2mL of 0.1M NaOH and 11.175g potassium chloride (KCl) were added, diluted to 1L and adjusted to pH 7]

Procedure: In a plastic sample cup, 5mL of chilled (0°C) Bendall solution was added to 1g of sample and homogenized for 15 seconds using an Ultra Turrax (13500rpm). The pH of the homogenate was measured using a pH meter where the temperature of measurement was set to 0°C. The measurement was done in duplicate.

D. Water Activity (Aw)

Material: Aqualab, Series 4TE Decagon Devices SN 540001787 Incubator at 22°C Plastic sample cups Raw anchovies samples or salt-fermented anchovy pastes

Procedure:

The Aw value was measured using the chilled mirror dewpoint technique at 25°C. Samples were thawed first and then incubated at 22°C for at least 30 minutes to lower the temperature difference between the sample and the instrument. Around 80% of the sample cup was filled with the sample which was then placed in the sealed chamber of the Aqualab equipment containing a mirror. When the mirror cooled down, a photoelectric cell detected the change in reflectance from the time when condensation occurred on the mirror. After the sample and the head-space of the sealed chamber reached the equilibrium, the relative humidity of the air in the chamber was measured, which also corresponds to the water activity of the sample. At equilibrium, the temperature was recorded and finally the Aw value was calculated by the Aqualab equipment using the vapor pressure chart. All samples were measured in duplicate.

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E. Mineral Content

Material: ICP-AES (Inductively Coupled Plasma - Atomic Emission Spectroscopy) – VARIAN VISTA-MPX Muffle oven Crucibles Filter paper Volumetric flasks Raw anchovies samples or salt-fermented anchovy pastes

Reagents: 70% Concentrated nitric acid (HNO3) Doubled distilled water (DDW) Blank (1% Nitric acid solution) ICP multi-element standard solution (1000mg/l) Working standards (10, 20, 50, 100, 200 mg/l standards prepared by using the ICP multi- element standard)

Procedure: In dried crucibles, 5 grams of samples were weighed. Samples were then burned using a Bunsen burner to eliminate most of the organic matter. Crucibles were then placed in the muffle oven at 600°C for at least 17 hours until white ashes were obtained. They were then taken out of the muffle oven and cooled down at room temperature. After cooling, samples were dissolved in 5mL nitric acid and filtered in a 10mL volumetric flask. Crucibles were washed with DDW and again poured on the filter. More DDW was added to the flask until the mark and this for the determination of microelements copper, iron, zinc and manganese. Samples were further diluted up to 1000 times for the determination of macroelements calcium, magnesium and potassium; and 2000 times for sodium. The blank, working standards and samples were then analyzed using ICP-AES containing a plasma and auxiliary flow of 15L/min and 1.5L/min respectively. The pump rate was 15rpm and the nebulizer pressure was set at 1kPa. Absorbance was measured for each element at 5 different wavelengths. Mineral content were calculated using the formula: (푐 ∗ 푑)/1000 퐶표푛푐 = 푚푎푐푟표 푉푠 ∗ 퐷푀

(푐 ∗ 푑) ∗ 1000/1000 퐶표푛푐 = 푚푖푐푟표 푉푠 ∗ 퐷푀

Where: Concmacro = concentration of each macroelement (mg/g DM)

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Concmicro = concentration of each microelement (µg/g DM) c = calculated concentration by ICP instrument (mg/L) d = dilution factor (mL) Vs = weight of the fresh sample (g) DM = dry matter content of sample (g/100g fresh sample)

F. Salt Content

Salt content (NaCl) was calculated from the concentration of sodium (Na+) obtained by mineral analysis with ICP-AES using the formula:

푎 푥 푏 푁푎퐶푙 = 푐

Where : NaCl = concentration of NaCl (g/kg dry matter sample) a = molar mass of NaCl (58.5 g/mol) b = concentration of Na (mg/g dry matter sample) c = molar mass of Na (23 g/mol)

G. Fatty Acid Composition a. Anchovy Paste Fatty Acids Extraction (Folch et al., 1957)

Material: Glass extraction tubes with screw caps Analytical balance 100mL Volumetric flasks Filters Test tubes with screw caps Centrifuge Jet filter pump Separatory funnels Rotavapor flasks Rotating evaporator Raw anchovies samples or salt-fermented anchovy pastes

Reagents: Chloroform/Methanol solution (C/M) – 2:1 v/v 0.1% Butylhydroxytoluene (BHT) in chloroform (1g BHT in 1L chloroform) Chloroform

26

Procedure: In an extraction tube, 5 grams of frozen sample was weighed to which 25mL C/M solution and 3mL of 0.1% BHT solution were added. Samples were homogenized for 1 minute using an Ultra Turrax (13500rpm). The bar of the Ultra Turrax was washed with 10mL C/M solution which was collected in the extraction tube. The homogenate was left overnight at room temperature in a dark area. In a 100mL volumetric flask, the homogenate was filtered and collected. The empty extraction tube, which contained the homogenate, was washed with 10mL C/M solution twice and poured on the same filter. The filter was rinsed after draining with 5mL C/M. After 10 minutes, the filtrate was divided into two test tubes containing 15mL of distilled water each. The volumetric flask was washed with 5mL C/M and the solution was placed in one of the 2 test tubes. Tubes were then centrifuged at 3000rpm for 10 minutes. Afterwards, the upper layer of the solution was removed by a water jet filter pump. The remaining content of both tubes was transferred to one separatory funnel with a glass funnel. The tubes were washed with 5mL C/M each. Two separated layers were noticeable after 20 minutes and the bottom layer was collected in a rotavapor flask. The glass funnel and inside of the separatory funnel were washed with 5mL C/M each. After 20 minutes, to obtain a separation, the bottom layer was again collected in the rotavapor flask. The extracts were evaporated with the rotating evaporator (water bath: 40°C, 100rpm). Subsequently, the fat was re-dissolved in 10mL chloroform and put in test tubes which were stored at -20°C.

b. Fatty acid Methylation (Raes et al., 2001)

Material: Test tubes Warm water bath at 50°C Vortex Centrifuge Tip tubes Pasteur pipette Evaporator under nitrogen

Reagents: 0.5N NaOH in methanol (20g NaOH dissolved in 1mL MeOH) HCl in MeOH (0.5L HCl mixed in 0.5L MeOH) Internal standard (IS) solution (2mg C19:0 per mL hexane)

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Procedure: Previously stored extracts were allowed to warm up to room temperature. Then 1mL extract and 1mL IS were placed in test tubes. The solution was evaporated under nitrogen after which 3mL of 0.5N NaOH/MeOH was added. After stirring the test tubes with a vortex, the samples were kept at 50°C for 30 minutes. Afterwards, 2 mL HCl/MeOH was added, vortex and then kept again at 50°C for 10 minutes. The tubes were shaken and cooled down to room temperature. Fatty Acid Methyl Esters (FAME) were extracted by the addition of 2mL hexane and 2mL distilled water to the cooled solution which was then stirred with a vortex and centrifuged at 2000rpm for 5 minutes. Using a Pasteur pipette, the upper layer was removed and transferred to a tip tube. Again, 2mL of hexane was added to the remaining solution which was then stirred with a vortex and centrifuged. The upper layer was removed again and transferred to the same tip tube by a Pasteur pipette. Hexane was evaporated under nitrogen and finally, FAME were re-dissolved in 1mL hexane and transferred to vials which were stored at -20°C.

The FAME were analyzed using Gas Chromatography with a column of HP88 Agilent (60mx0.25mmx0.2µm) and FID detector. The injector and detector temperature, flow rate over column and injection volume were 250°C, 280°C, 2mL/min and 1µL respectively. The initial temperature of the column was 120°C which was held for 1 minute and then increased to 175°C at a rate of 6°C/min. The temperature was again increased to 210°C for 6.5 minutes which was further increased at a rate of 5°C/min to 230°C. Holding time at 230°C was 5 minutes. Fatty acid proportion was determined by the formula:

푋 푃푟표푝 = ( ) ∗ 100 푌

Where: Prop = proportion of each fatty acid (g/100g FAME) X = peak area of the specific fatty acid Y = total amount of area of known and unknown fatty acids (excluding BHT and IS)

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H. Total and Free Non-Protein Nitrogen Content (Oddy, 1974)

Material: Ultra Turrax Filter paper Boiling water bath Drying oven at 103±2°C Spectrophotometer Sample cups Raw anchovies samples or salt-fermented anchovy pastes

Reagents: 0.6M Perchloric acid (HClO4) [103mL concentrated HClO4 (70%) diluted to 2L] 8.4M HCl solution 15M NaOH solution 2M NaOH solution Buffer solution pH 5.8 [mixture of 250mL propionic acid (C3H6O2), 250mL of 2- methoxyethanol (C3H8O2), 350mL distilled water, and 100mL 15M NaOH, diluted to 1L] Ninhydrin solution [5g ninhydrin (C9H6O4) dissolved in 1L buffer solution] Reductant solution [50mg ascorbic acid (C6H8O6) dissolved in 50ml distilled water] Leucine stock solution [74.8mg L-leucine (C6H13NO2) dissolved in 100mL distilled water] Standard series [0.5, 1.0, 2.0, 3.0, 4.0 and 5.0mL of stock solution diluted to 100mL distilled water] 60% ethanol solution

a. Perchloric Acid Extraction of Anchovy Paste Samples

Procedure: In a sample cup, 5g of sample with 40mL of 0.6M perchloric acid (PCA) were homogenized using an Ultra Turrax at 8000rpm for 1 minute. The bar of the Ultra Turrax was washed with 10mL of PCA which was collected in the sample cup. The suspension was filtered with filter paper over a 100mL volumetric flask and the container was rinsed with 30mL PCA which was again filtered over the flask. PCA was added until the mark. The extracted samples were stored at -20°C for free and total non-protein nitrogen analysis.

29 b. Total Non-Protein Nitrogen

Procedure: From each sample, 2 mL of PCA extract was transferred to a test tube to which 5mL of 8.4M HCl was added. Then the solution was mixed, covered and put in the oven (103°C) for 24 hours. After hydrolysis, the samples were neutralized with 2M NaOH to attain a pH between 4 and 10. Samples were then transferred to 50mL volumetric flask and then diluted with distilled water until the mark. From each sample, 1mL of diluted (100 times with buffer solution) hydrolyzed PCA extract was transferred in duplicate to test tubes. From each standard solution, 1 ml was transferred to test tubes. Distilled water was used as a blank. To each test tube, 100µL reductant solution and 1mL of ninhydrin solution were added after which solutions were stirred using a vortex. Tubes were covered with aluminum foil and placed in a boiling warm water bath for 20 minutes. After cooling down the tubes with tap water, 5mL of ethanol solution was added, after which samples were stirred using a vortex. After 15 minutes, the mixture was stirred again and the absorbance was measured at 570nm. Total NPN was calculated using the following formula:

푇표푡푎푙푁푃푁 = 푥∗푑∗50∗ 100∗100 푉푠∗퐷푀∗1000 Where: Total NPN = total α-NH2-N content in dry matter (mg/100gDM) X = amount of total α-NH2-N obtained from a calibration curve (µg/mL) d = dilution factor (mL) Vs = weight of fresh sample used in PCA extract (g) DM = dry matter (gDM/100g fresh sample)

c. Free Non-Protein Nitrogen

Procedure: Free NPN was analyzed on the same way as the total NPN except for the hydrolysis part. From each sample, 1mL of diluted (100 times with buffer solution) PCA extract was transferred in duplicate to test tubes. From each standard solution, 1 ml was transferred to test tubes. Distilled water was used as a blank. To each test tube, 100µL reductant solution and 1mL of ninhydrin solution were added after which samples were stirred using a vortex. Tubes were covered with aluminum foil placed in a boiling warm water bath for 20 minutes. After cooling

30 down with tap water, 5mL of ethanol solution was added and samples were stirred using a vortex. After 15 minutes, the mixture was stirred again and the absorbance was measured at 570nm. Free NPN was calculated using the following formula:

퐹푟푒푒푁푃푁 = 푥∗푑∗100∗100 푉푠∗퐷푀∗1000

Where: Free NPN = free α-NH2-N content in dry matter (mg/100gDM) X = amount of free α-NH2-N obtained from calibration curve (µg/mL) d = dilution factor (mL) Vs = weight of fresh sample used in PCA extract (g) DM = dry matter (gDM/100g fresh sample)

I. TBARS Analysis (Tarladgis, et al., 1964)

Material: Distillation apparatus (tubes and distillation unit) Ultra Turrax Spectrophotometer Boiling warm water bath 100mL Scott bottle 100mL Volumetric flask Raw anchovies samples or salt-fermented anchovy pastes

Reagents: TBA reagent [865 mg 2-thiobarbituric acid placed in a 100mL volumetric flask, covered with aluminum foil, was dissolved with 75mL of acetic acid in a hot plate at 70°C. After cooling down at room temperature, 2mL of concentrated HCl (37%) was added and filled up to the mark with acetic acid.] BHT solution [1.5g of 2,6-di-tertiary-butyl-4-methylphenol dissolved with ethanol in a 100mL volumetric flask and stored in a dark bottle at room temperature] 4M HCl solution Standard stock solution [14.4µL of 1,1,3,3-tetramethoxypropane dissolved in 100mL distilled water] Standard working solution [20 times dilution of the stock solution (1mL standard stock solution dissolved in 20mL distilled water)]

31

Standard series : concentration (nmol/5mL) µL working solution mL water (blank) 0.0 0.0 5.00 2.19 50 4.95 4.37 100 4.90 8.74 200 4.80 13.12 300 4.70 17.49 400 4.60 21.86 500 4.50

Procedure: In a 100mL scott bottle, 10g of sample was weighed and then 40mL distilled water and 1mL BHT solution was added. The sample was homogenized using an Ultra Turrax (13000rpm) for 30 seconds and then transferred to the distillation tube. The bottle was again added with 30mL of distilled water and homogenized for a while with Ultra Turrax to rinse its dispersing unit, and then transferred again to the distillation tube. To the distillation tube containing the homogenate, 3mL of 4M HCl was added. After cleaning the distillation unit with distilled water, the tube was attached. Distillate was collected in a 100mL volumetric flask until the mark. From the collected distillate, 5mL was transferred to test tubes in duplicate. Also, 5ml standard solutions were transferred in tubes. To the tubes with sample distillates, or with standard solutions, 1mL TBA reagent was added, then stirred in a vortex and put in a boiling warm water bath for 35 minutes. After cooling down the tubes at room temperature with tap water, the absorbance was measured at 532nm. Samples were diluted 3 times before the absorbance was read. The concentration of TBARS was calculated with the formula:

푇퐵퐴푅푆 = 푐 푥 20 푥 푑 푥 72 푚 푥 퐷푀∗1000

Where: TBARS = TBARS content (µg malonaldehyde/gDM) c = calculated concentration of TBARS obtained from the calibration curve (nmol malonaldehyde/5mL distillate of sample) d = dilution factor (mL) 72 = molar mass of malonaldehyde (µg/µmol) m = weight of fresh sample (g) DM = dry matter (gDM/100g fresh sample)

32

J. Microbiological Analysis

Materials: Crushed ice 8mL sterile Glycerol 10mL sterile Brain Heart Infusion (BHI) medium 9mL physiological water Plate Count Agar (PCA) de Man, Rogosa Sharpe (MRS) Agar (MRS Broth added with Bacteriological Agar) Nutrient Agar (Nutrient Broth added with Bacteriological Agar) Malt Extract Agar (Malt Extract Broth added with Bacteriological Agar) Violet Red Bile Glucose Agar (VRBGA) Sodium Chloride (NaCl) Potassium Sorbate Casein Incubators Test tubes Plates Drigalski spatula Ethanol

Procedure: a. Sampling:

From the liquid part of the fermented fish products collected, 2mL was taken and placed into falcon tubes containing 8mL glycerol. These glycerol stocks were transported from the Philippines to Belgium with the same procedure done with the collected fermented anchovy pastes and stored at -20°C upon arrival at UGent Kortrijk Campus laboratory until analyzed.

b. Microbial Analysis

Glycerol stock was thawed under chilled condition with crushed ice until liquid becomes less viscous. The glycerol stock was vortex and 250µL of the glycerol stock was transferred to BHI medium, and then incubated for 4 hours at 30°C. Dilution series until 10-5 of the BHI- cultured broth was made. From the cultured broth (100) and dilutions 10-1, 10-3 and 10-5, 0.1mL was taken and plated (spread plate) on the different agar plates. Total aerobic plate count was analyzed using PCA and incubated for 48 hours at 37°C, while for the total halophilic plate count PCA with 10% NaCl was used and incubated for 14 days at 37°C. For the total lactic acid

33 bacteria, MRS agar with 1.4g/L potassium sorbate was used and incubated for 72 hours at 30°C. Total halophilic lactic acid bacteria was analyzed using the former medium added with 10% NaCl and also incubated for 72 hours at 30°C. For the total proteolytic bacteria, nutrient agar supplemented with 10% NaCl and 1% casein was used and incubated at 37°C for 48 hours. Halophilic yeast counts were analyzed on Malt extract agar added with 10% NaCl, and at pH of 4.8 and incubated at 25°C for 72 hours. Total enterobacteriaceae were analyzed with VRBA and incubated for 24 hours at 37°C. Media used were sterilized first before putting into plates except for the VRBA. All the visible colonies were counted and represented as colony forming units (CFU). The number of microorganisms were calculated using the formula:

퐴 푥 푉 푋 = 푙

Where : X = number of microorganisms (CFU/mL) A = number of colonies on the plate (CFU) V = reciprocal of the dilution factor l = volume of inoculum (mL)

K. Statistical Analysis

Data were analyzed statistically using SPSS Statistics 21 software (IBM Corporation, 2012). Normality of data distribution and equality of variance were considered using Kolmogorov-Smirnov and Modified Levene tests. One-way ANOVA (parametric) and Kruskal Wallis (non-parametric) tests were used for hypothesis testing. Significant differences between the results from the different time points and producers of anchovy paste were determined. In conjunction with ANOVA, Tukey test was used to find which means were significantly different from each other. All the used statistical tools were set at 5% level of significance.

34

Chapter 4. Results and Discussion

A. Raw materials

Salt-fermented anchovy paste samples were traditionally produced. Three different batches from one local producer (SP) were sampled for different time points (T) of fermentation (raw fish =R; day0=D0; day9=D9; day19=D19; day28=D28) (figure 2). Three locally salt- fermented anchovy pastes, sold in markets (FP) were also purchased (figure 3). Descriptions of the samples are presented in table 9. Names of the producers and their years of experience in making bagoong are also included.

Table 9. Characterization of the samples and manufacturers

Date/Time Other Name of Manufacturer Region of Origin Manufactured Characteristics  1:3.6 salt:fish ratio  fish used was SPA. Gimaylan Salted Fish caught on the July 2013 Processors Organization previous day and 8AM (6 years experience) stored at -10°C before processing  unwashed fish  liquid drained  1:3.6 salt:fish Misamis Oriental ratio SPB. Gimaylan Salted Fish  used freshly July 2013 Processors Organization caught fish from 10AM (6 years experience) local fishermen  unwashed fish  liquid drained  1:3.6 salt:fish ratio SPC. Gimaylan Salted Fish  fish used from July 2013 Processors Organization earlier catch of 1PM (6 years experience) local fishermen  unwashed fish  liquid drained FPA. Escalona May 2013 Surigao del Norte (20 years experience)  salt and fish ratio FPB. Millan July 2013 Surigao del Sur not specified (5 years experience)

FPC. Durando April 2013 Surigao del Sur (17 years experience)

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Figure 2. Fermented anchovy pastes at different time points

Figure 3. Fermented anchovy pastes locally sold in markets

36

B. Biochemical and chemical composition of commercially available anchovy pastes

a. Chemical Composition

Gross composition of the different purchased anchovy pastes are given in table 10. Dry matter content of FP varied between 36-40g/100g and no significant differences between samples were observed (p˃0.05). The DM content of the samples is slightly higher than the typical anchovy paste (bagoong dilis) mentioned by Chinte-Sanchez (2008) which has a value of 32.9g/100g. It is also within the range of fish pastes reported by Montaño, et al. (2001) from dilis available in a main public market in the northern part of the Philippines, which has a mean value of 39.2g/100g, and within the Philippine Standards for bagoong which was 40g/100g (Chinte- Sanchez, 2008). However, it has lower values compared to nga-pi of Myanmar which is 60g/100g (Tyn, 1993), terasi and pedah of Indonesia which is 50-65 and 53-56g/100g respectively (Putro, 1993; Aryanta, 2000), and belacan of Malaysia which has 60-73g/100g of DM content.

Crude fat content of FP varied from 4-6g/100gDM which is similar to what Chinte- Sanchez (2008) has stated with the value of 5.78g/100gDM. Also, it has higher levels than the one obtained by Montaño, et al. (2001) which has 1.53g/100gDM, fish pastes from Myanmar containing 2.5g/100gDM (Tyn, 1993), and fish paste from Malaysia with 1.9-4.3g/100gDM of fats (Abdul Karim, 1993). However, it has lower values compared to pedah which is 20.86g/100gDM (Putro, 1993) but just of the same range with terasi from Indonesia which has a value of 2.5-6.4g/100gDM (Aryanta, 2000). The analyzed fat content of FP showed no significant differences (p˃0.05) between the different producers.

There were also no significant differences (p˃0.05) observed between crude protein of FP, varying between 34-44g/100gDM. These levels are a bit higher to the Philippine Standards for Bagoong which is 31.25g/100gDM, and as mentioned by Chinte-Sanchez (2008) which is 31.31g/100gDM. It is also higher than nga-pi (30g/100gDM) of Myanmar (Tyn, 1993). On the other hand, these levels are comparable with terasi and belacan of Indonesia and Malaysia (31- 69 and 39.3-66.7g/100gDM respectively) (Aryanta, 2000; Abdul Karim, 1993).

37

Table 10. Chemical composition of anchovy paste traditionally produced in some parts of the Philippines

Sample

FPA FPB FPC p-value DM (g/100g) 35.9±0.54 40.4±1.33 40.2±1.57 0.060

Crude Fat (g/100gDM) 6.47±1.92 4.55±0.38 4.45±0.05 0.240

Crude Protein (g/100gDM) 39.4±1.45 43.8±4.3 34.4±4.58 0.180

Aw 0.80±0.001 0.75±0.002 0.74±0.002 0.102 pH* 5.33±0.11a 6.39±0.02b 6.55±0.14c 0.007

NaCl (g/100gDM) 37.0±10.8 46.3±3.28 34.6±4.29 0.341

Results expressed as mean ± standard deviation; n=2;*-n=4 Values within the row with the different superscripts (a-c) denote significant difference from each other, p ≤ 0.05.

Other parameters analyzed were water activity (Aw), NaCl content and pH (table 10). The

Aw of FP ranged between 0.74-0.80. It coincides to the studies of Sumino, et al., (1999,2003) on

bagoong which has an average Aw of 0.77 and belacan of Malaysia with an average Aw of 0.74.

On the other hand, terasi of Indonesia and kapi of Thailand got an average Aw of 0.718 and 0.71

respectively(Sumino, et al., 1999). There were no significant differences (p˃0.05) in Aw for the different producers of FP. NaCl content of FP varied between 34-46g/100gDM and no significant differences (p˃0.05) were observed between different producers. These values were lower than the one obtained by Montaño, et al., (2001) which is 59.69g/100gDM, and the Philippine Standards for Bagoong which is 50-62.5g/100gDM (Chinte-Sanchez, 2008). However, the analyzed salt content is in agreement with terasi of Indonesia as reported by Aryanta (2000) which contains 23-40g/100gDM, belacan of Malaysia (Abdul Karim, 1993) containing 18-42g/100gDM, and nga-pi of Myanmar with 42g/100gDM of salt (Tyn, 1993). The pH of FP varied from 5.3-6.6 and was significantly different (p<0.05) from each other. It is in agreement with budu (fish sauce from anchovies) of Malaysia which has a pH of 5.4-6.2 (Abdul Karim, 1993). In contrast, the belacan (from shrimp) of Malaysia and terasi (from fish and/or shrimp) of Indonesia has a rather neutral pH of 7.2-7.6 and 7.5 respectively (Abdul Karim, 1993; Surono & Hosono, 1994). The pH of fermented fish varies depending on the biological

38 properties of fish. Furthermore, different species of fish affects its biological properties resulting to production of different kinds of peptides and amino acids (Ng, et al., 2011). This could be the reason why the reported pH values of belacan and terasi differs with the sampled anchovy pastes. On the other hand, budu, which is a fish sauce, acquired same pH level due to the fact that it was also produced from anchovies. The pH of FP, though from the same raw materials, also varies from each other because of their difference in the ripening period. FPA, which is 3- month old, has lower pH than FPB which is fermented only for 1 month. The decrease in pH is due to the production of acids (Kilinc, et al., 2005; Ng, et al., 2011). Moreover, as fermentation continues and protein hydrolysis starts, proteins are degraded resulting to increase in pH due to the production of N-compounds (Kilinc, et al., 2005; Ng, et al., 2011). Such N-compounds produced are ammonia and trimethylamine (TMA) which have basic properties that will react with acidic compounds forming a more stable compound (Ng, et al., 2011).

b. Mineral Composition

Mineral content of the product is shown in table 11. Minerals are divided into micro- and macroelements. In this study, microelements analyzed include copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn). On the other hand, macroelements analyzed were magnesium (Mg), potassium (K), calcium (Ca) and sodium (Na). Microelements analyzed resulted to non- detectable values for Cu and Mn while Fe and Zn varied between 15.8-18.9 and 5-28µg/gDM respectively. No significant difference (p˃0.05) was observed in Fe while Zn content of FPA was significantly lower compared to FPB and FPC (p-value<0.05). For the macroelements, values obtained were between 1.25-11, 12-16, 36-68 and 136-182mg/gDM for Mg, K, Ca and Na respectively. There was no significant difference (p˃0.05) between producers for K and Na analyzed. Whereas, Mg of the different FP varies significantly from each other (p-value<0.05) and Ca of FPA differs significantly (p-value<0.05) from FPC. The amount of Ca in this study is higher than the one reported by Chinte-Sanchez (2008) whose value was 16.26g/kgDM for bagoong dilis. The value of K for FP (500-600mg/100g) is higher than those studied by Sumino, et al. (1999, 2003) which had a mean K content of 157±30mg/100g in bagoong, 466±144mg/100g in terasi, 116mg/100g in kapi and 207mg/100g in belacan. According to Chinte-Sanchez (2008), bagoong dilis have an Fe level of 331.31µg/gDM which is much higher

39 than the ones obtained in this study. Also the Zn in the analyzed FP (1.52-11.7mg/kg) is higher than the shrimp pastes obtained by (Pilapil, et al., 2015) whose values were 5.94-6.92mg/kg. The difference in the mineral content of the studied anchovy pastes from other studies is due to the fact that composition of fish varies with regards to their diet, feed rate, genetic strain, age, size, sex, sexual maturity, and seasonal differences (Sankar, et al., 2013; Bakhiet, et al., 2013). Also, FP attained a high Na content due to the added salt for the fermentation to take place.

Table 11. Mineral composition of anchovy paste traditionally produced in some parts of the Philippines

Sample Mineral FPA FPB FPC p-value Cu ND ND ND

Microelement Fe 18.9±6.25 ND 15.8±5.14 0.643 (μg/g DM) Mn ND ND ND

Zn 5.39±1.71a 28.1±0.39b 20.9±6.01b 0.018 Mg 1.25±0.003a 1.95±0.18b 11±1.38c <0.001

Macroelement K 15.9±10.03 14.9±2.0 12.5±1.56 0.215 (mg/g DM) Ca 36.4±1.69a 49.5±6.91ab 67.7±8.58b 0.037 Na 146±42.35 182±12.88 136±16.86 0.341 Results expressed as mean ± standard deviation; n=2. ND=below the detection limit. Values within the row with the different superscripts (a-c) denote significant difference from each other, p ≤ 0.05.

c. Fatty Acid Composition

Fatty acids can be saturated and unsaturated. Saturated fatty acids (SFA) contain no double bond in their carbon chain, while unsaturated fatty acids have double bonds in their carbon chain. Unsaturated fatty acids are classified as monounsaturated (MUFA) and polyunsaturated (PUFA) containing only one double bond and two or more double bonds respectively. In tables 12 and 13, SFA present in the analyzed anchovy pastes are lauric acid (C12:0), tridecanoic acid (C13:0), myristic acid (C14:0), pentadecanoic acid (C15:0), palmitic

40 acid (C16:0), margaric acid (C17:0), stearic acid (C18:0), arachidic acid (C20:0), heneicosanoic acid (C21:0), and behenic acid (C22:0). MUFA consists of myristoleic acid (C14:1), 10- pentadecenoic acid (C15:1), palmitoleic acid (C16:1), 10-heptadecenoic acid (C17:1), oleic acid (C18:1), and gadoleic acid (C20:1). Linoleic acid (C18:2c), γ-linolenic acid (C18:3n-6), α- linolenic acid (C18:3ω-3), 11,14-eicosadienoic acid (C20:2), arachidonic acid (C20:4), eicosapentaenoic acid (C20:5ω-3; EPA), docosapentaenoic acid (C22:5ω-3), and docosahexaenoic acid (C22:6ω-3; DHA) are among the PUFA.

Total SFA, MUFA and PUFA obtained from FP ranges from 44.5-53.4, 14-18, and 18.9- 31.3g/100g FAME respectively. These fatty acids differ significantly (p-value<0.05) where FPB differs from FPA and FPC. According to Montaño, et al. (2001) and Peralta, et al. (2008), long periods of fermentation doesn’t affect the highly unsaturated fatty acids such as EPA and DHA. But, it can be observed from this study that with longer fermentation periods, SFA and MUFA decreases while PUFA increases. However, the differences in FP could not be clearly identified due to lack of information towards how these products are processed. Table 13 shows the fatty acid profile of raw and different anchovy pastes obtained from different studies. The SFA content of FP obtained is concurrent with the results of Sumino, et al. (1999, 2003) for bagoong,belacan and kapi, however, the one reported by Montaño, et al. (2001) has a higher value. It can also be observed that the raw anchovy obtained by Sankar, et al. (2013) has a lower SFA value than the fermented anchovies. For MUFA, FP is in agreement with bagoong reported by Sumino, et al. (1999) but a little bit lower than the studies of Montaño, et al. (2001), belacan and kapi of Sumino, et al.(1999,2003) and raw anchovy from Sankar, et al. (2013). The PUFA content of FP is higher compared to the one reported by Montaño, et al. (2001) but lower than the bagoong and belacan studied by Sumino, et al. (1999,2003) and raw anchovy by Sankar, et al. (2013). However, it is just in agreement with the PUFA of kapi reported by Sumino, et al. (1999).

PUFA can be divided into omega3 (ω3) and omega6 (ω6) fatty acids. Total ω3 obtained in this study range a value between 14.6-26.9g/100g FAME which is higher than the one obtained from the study of Montaño, et al. (2001) which has 4.7g/100gFAME. It is in agreement with kapi and belacan of Thailand and Malaysia (Sumino, et al., 1999; Sumino, et al., 2003) but

41 lower than the bagoong from the Philippines obtained by Sumino, et al. (1999) (Table 13). Total ω6 of FP varies from 4.31-4.49g/100gFAME and these results coincide with kapi studied by Sumino, et al (1999). They are also higher than the ones obtained by Montaño (2001) and belacan and bagoong of Sumino, et al (1999,2003) (Table 13). Total ω6 shows no significant difference (p-value˃0.05) from the different producers while total ω3 differs significantly (p- value<0.05) where FPB has lower value than FPA and FPC.

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Table 12. Fatty acid profile (g/100g FAME) of anchovy paste traditionally produced in some parts of the Philippines

Sample FAME (g/100g) FPA FPB FPC p-value C 12:0 0.17±0.003 0.23±0.02 1.17±0.28 0.102 C 13:0 0.08±0.00 0.17±0.01 0.17±0.04 0.063 C 14:0 5.42±0.07a 6.75±0.13b 5.38±0.34ac 0.012 C 15:0 1.36±0.03 2.08±0.04 1.35±0.06 0.180 C 16:0 26.4±0.73a 27.9±0.26ab 23.5±1.04ac 0.022 C 17:0 2.42±0.08a 2.63±0.009ab 2.26±0.12ac 0.046 C 18:0 8.35±0.07a 12.94±0.3b 10.1±0.26c 0.001 C 20:0 0.31±0.004a 0.23±0.002b 0.25±0.02b 0.007 C21:0 0.1±0.004 0.22±0.19 0.09±0.006 0.368 C22:0 0.15±0.008 0.24±0.01 0.22±0.04 0.076

C 14:1 0.21±0.002 0.32±0.2 0.31±0.02 0.643 C 15:1 0.27±0.03a 0.34±0.005ab 0.54±0.04c 0.005 C 16:1 4.48±0.14a 7.13±0.45b 3.82±0.22ac 0.003 C 17:1 0.85±0.09a 1.28±0.02b 0.88±0.02ac 0.013 C 18:1 8.1±0.18a 8.8±0.05ab 7.68±0.31ac 0.028 C20:1 1.1±0.02a 0.49±0.02b 0.72±0.07c 0.002

C 18:2ω-6 1.73±0.05 1.51±0.1 1.54±0.15 0.245 C 18:3ω-6 0.41±0.06 0.45±0.009 0.51±0.2 0.798 C20:2 ω-6 0.15±0.03 0.4±0.06 0.32±0.13 0.111 C20:4 ω-6 2.07±0.02 1.95±0.1 2.12±0.09 0.230

C18:3ω-3 0.9±0.02a 0.68±0.07b 0.85±0.04ab 0.045 C20:5ω-3 6.09±0.06a 2.77±0.16b 4.38±0.35c 0.002 C22:5ω-3 1.35±0.2 0.98±0.33 1.67±0.38 0.233 C22:6ω-3 18.6±0.04a 10.2±0.89b 16.7±0.61ac 0.002

Total SFA 44.8±1.00a 53.4±0.98b 44.5±2.2ac 0.012 Total MUFA 15±0.46a 18.4±0.74b 14±0.69ac 0.009 Total PUFA 31.3±0.47a 18.9±1.73b 28.1±1.92ac 0.003 Total ω3 26.9±0.28a 14.6±1.46b 23.6±0.63ac 0.002 Total ω6 4.36±0.16 4.31±0.269 4.49±0.57 0.669 Results expressed as mean ± standard deviation; n=2. Values within the row with the different superscripts (a-c) denote significant difference from each other, p ≤ 0.05.

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Table 13. Fatty acid profile of anchovy pastes obtained from different studies

Bagoong Montaño Sankar, et al., Belacan (Sumino, Kapi (Sumino, FAME (g/100g) (Sumino, et al., (2001) (2013)* et al., 2003) et al., 1999) 1999) C 12:0 1.3±1.1 0.03±0 nd nd nd C 13:0 nd 0.08±0 nd nd nd C 14:0 11.3±0.2 1.8±0.11 4.2±1.1 6.0±3.7 5.7±1.7 C 15:0 nd 1.29±0.12 nd nd nd C 16:0 38.4±1.5 8.79±0.34 30.3±2.0 33.4±6.0 26.2±5.3 C 17:0 nd 1.06±0.3 nd nd nd C 18:0 16.6±0.2 8.09±0.45 10.3±2.2 13.0±2.1 10.2±1.2 C 20:0 1.08±0.07 nd nd nd nd C 21:0 nd nd nd nd nd C 22:0 0.9±0.1 nd nd nd nd

C 14:1 nd nd nd nd nd C 15:1 nd nd nd nd nd C 16:1 8.3±0.2 3.96±0.04 8.6±2.0 7.7±1.7 4.4±1.6 C 17:1 nd 0.91±0.07 nd nd nd C 18:1 12.3±0.4 8.94±0.56 13.0±6.3 13.3±4.0 8.3±2.1 C 20:1 1.0±0.2 6.08±0.45 nd nd 3.4±0.9

C 20:4 0.50±0.17 2.12±0.33 nd nd nd

C 18:3ω-3 0.14±0.12 2.32±0.15 nd nd nd C 20:5ω-3 0.5±0.4 4.97±0.39 14.3±3.4 7.9±3.0 9.3±2.7 C 22:5ω-3 0.05±0.08 nd nd nd nd C 22:6ω-3 1.7±0.2 22.5±1.15 10.7±2.9 8.9±4.3 30.3±6.8

C 18:2ω-6 0.63±0.03 0.45±0.04 2.9±3.9 4.4±1.7 1.5±0.4 C 18:3ω-6 nd nd nd nd nd C 20:2ω-6 0.12±0.11 12±0.56 nd nd nd

Total SFA 71.5±2.0 24±1.2 44.8±5.3 52.4±11.8 42.1±8.2 Total MUFA 21.3±0.5 23.9±1.09 21.6±8.3 23.9±6.6 16.1±4.6 Total PUFA 7.3±1.8 51.9±1.99 33.5±11.8 23.7±10.3 41.8±10.6 Total ω3 4.7±0.9 29.8 25±6.3 16.8±7.3 39.6±9.5 Total ω6 2.6±0.9 15.9 2.9±3.9 4.4±1.7 1.5±0.4 Results expressed as mean ± standard deviation. nd=not determined. * = unfermented

44

d. Non-protein and TBARS composition

Non-protein nitrogen (NPN) content of the anchovy pastes was also analyzed (table 14). NPN are products of protein hydrolysis which includes peptides, free amino acids, amines and ammonia, which are responsible for the flavor and aroma development of fermented products (Jiang, et al., 2007; Majumdar & Basu, 2010; Kim, et al., 2003; Peralta, et al., 2008). Total NPN includes all the substances produced during the 24-hour acid hydrolysis while free NPN concerns the α-amino nitrogen readily present (not peptide-bound) in the product. Total NPN of FP varied between 76-107mg/100gDM). This is lower compared to the fermented fish reported by Majumdar & Basu (2010) which has a total NPN content of 540mg/100gDM. Even though that there was no significant differences (p˃0.05) between producers, it can be observed that FPB has higher total NPN than FPA and FPC which is of the same pattern acquired with their crude protein content from table 10. This high value for NPN may have contributed to the high pH value of FPB (6.39). High pH of FPC (6.55) may be attributed to other factors than NPN. Free NPN content of FP varied between 12-20mg/100gDM where FPA is significantly higher (p<0.05) than FPB and FPC. The difference between producers might not be clear as the process of production of these fermented products is also unclear, but possible reasons could be due to their salt:fish ratio and to whether the raw fish have been washed or unwashed prior to processing. Salt affects protein hydrolysis where the higher salt concentration, the faster is the proteolytic activity. This is in contrast to the study of Kim, et al. (2003) where lower salt concentration results to a higher hydrolyzing activities of proteases and peptidases in shrimp byproducts. According to the study of Giri, et al. (2009), washing step removed the extractive nitrogen resulting to a lower free NPN content. Aside from that, levels of extractable nitrogen increases rapidly during fermentation period for unwashed fish due to oxidation and higher decomposition rate of protein. The free NPN content of the fermented fish in this study is still lower than the ones obtained by Majumdar & Basu (2010) which has 163mg/100gDM free NPN content. The difference in NPN content of FP to the other study is due to variability in raw materials used, production process and maturation period of the fermentation process (Faithong, et al., 2010). The NPN content increases with fermentation period (Majumdar, et al., 2006; Kilinc, et al., 2005). In the study of Majumdar & Basu (2010), Indian shad was used and fermentation period is 4-6 months thus, acquiring a higher level of NPN.

45

Another chemical parameter analyzed in this study was thiobarbituric acid reactive substances (TBARS). TBARS is used as an indicator of lipid oxidation in meat and fish products (Irwin & Hedges, 2004). Values of TBARS analyzed for FP (table 14) ranged from 10.33- 21.73µgMDA/gDM which showed significant difference (p<0.05) between producers. FPC, which has a maturation period of 4 months, has a lower value than FPA and FPB with ripening periods of 3 and 1 month respectively. It can be observed that there is a decreasing trend of TBA with fermentation period, however, FPA and FPB doesn’t differ significantly from each other. According to Peralta, et al. (2008), prolonging the fermentation period results in the production of substances that contribute to increase in antioxidant activity and the ability to suppress lipid oxidation, thus, lowering the TBA value. This would explain why FPB have higher TBARS values compared to FPA and FPC since FPB was just fermented for 1 month. Comparing FPA and FPC which has longer ripening period, FPA attain a higher TBARS content. This is attributed to its higher content of PUFA and Fe. PUFAs are highly susceptible to lipid oxidation (Rashid, et al., 1992; Binsas, et al., 2008) while copper acts as prooxidant (Ladikos & Lougovois, 1990).

Table 14. Non-protein nitrogen and TBARS of anchovy paste traditionally produced in some parts of the Philippines

Sample Composition A B C p-value Free NPN (mg/100gDM) 20.2±0.25a 12.4±4.04b 16.9±0.74b 0.018

Total NPN (mg/100gDM) 79.3±4.06 107±42 76.9±10.92 0.397

TBARS (µgMDA/gDM) 18.8±0.42a 20±1.95ab 13.2±3.26c 0.025 Results expressed as mean ± standard deviation; n=4. Values within the row with the different superscripts (a-c) denote significant difference from each other, p ≤ 0.05.

46

C. Characterization of fermented anchovy pastes

Salt-fermented anchovy pastes from different batches produced by a local producer (SP) were analyzed as a function of time (T). The sampled SPA, SPB and SPC showed no significant difference (p-value˃0.05) with each other in the different time points for all measured parameters. Therefore, the following analysis is done where the three sampled anchovy pastes are used as replicates.

a. Chemical Characterization

i. Chemical composition

The chemical composition of SP as a function of T is shown in table 15. Dry matter (DM) content of SP varied between 22-48g/100g. There was significant difference (p<0.05) between the raw fish to the different days of fermentation. It can be observed that DM increased from R to D19. A raw fish contains high amount of moisture as moisture is one of its major components thus, having a low DM (Giri, et al., 2009). The moment when the salt was added and then drained (D0), the free water in fish is removed through the draining. As fermentation goes on, more water is removed through osmosis by the action of the salt (Majumdar, et al., 2006; Petrus, et al., 2013; Chinte-Sanchez, 2008).

Crude fat content varied from 4.1-8.3g/100gDM for SP. The analyzed pastes showed no significant differences (p˃0.05) between T. This shows that the fat content is hardly affected by the fermentation process. But comparing the start and end of fermentation (D0 to D28), a decrease in fat content is attained which is consistent with the study of Kilinc, et al., (2005) on fish sauce processing. Petrus, et al., (2013) also cited a decrease in fat content during fermentation due to the leaching process of fish muscle in correlation with salt penetration.

Analyzed values of crude protein in SP ranged between 42.5-93.1g/100gDM, in which R significantly differed from the other T (p < 0.05). Fish is rich and a good source of protein, thus, having a high value of protein in R of SP (Kristinsson & Rasco, 2000). At D0, protein decreases

47 drastically due to the draining where soluble proteins are also removed together with the water. As fermentation goes on, protein also decreases due to protein hydrolysis and leaching out in the brine (El-Sebaiy & Metwalli, 1989).

Other parameters analyzed were water activity (Aw), pH and NaCl content (table 15).

The Aw of SP ranged between 0.82-0.99. There was no significant difference (p˃0.05) in T. There may not differ significantly with the statistical analysis, but it can be observed that there was a decrease in Aw from R to D0 and then a constant Aw to the proceeding T. The decrease in

Aw is attributed to the removal of moisture and the addition of salt which has an osmotic effect on the product. A decrease in moisture is accompanied with the increase in salt and ash content (El-Sebaiy & Metwalli, 1989; Majumdar, et al., 2006; Petrus, et al., 2013). NaCl content of SP ranged between 5.8-37.1g/gDM which has significant difference (p<0.05) between R and D0. Salt content increases due to the addition of NaCl to start the fermentation. Salt content began to increase up to D9, a decrease on D19 and then an increase afterwards. But comparing the beginning and end of fermentation in this study (D0 and D28), the result showed an increase in the salt content. This is because of osmosis where moisture is removed in replace of salt that has been absorbed into the fish flesh (Majumdar, et al., 2006; Petrus, et al., 2013). High salt content enhances the shelf-life of the fermented fish due to lower Aw, inhibiting the growth of spoilage microorganisms (Chinte-Sanchez, 2008; Majumdar & Basu, 2010; Montaño, et al., 2001). For pH of SP, analyzed values varied from 6.7-7.0 where there was no significant differences (p˃0.05) between T. This is in contrast to the study of Surono & Hosono (1994) on fermented fish product terasi where the pH level rises from 6.0 to 6.5 and later reduces to 4.5. Upon further fermentation, the pH increased to 7.8. It has been said that organic acids, such as lactic acid, are produced during fermentation resulting to lowering the pH. Moreover, protein degradation occurs during fermentation which releases nitrogenous compounds, such as amines and ammonia resulting to an increase in pH (Ng, et al., 2011; Kilinc, et al., 2005; Surono & Hosono, 1994). In the present study, there’s only a minimal change in pH and it is of a rather neutral pH. This result is similar to the one reported by Sarojnalini & Suchitra (2009) on starter fermented ‘Ngari’ with a pH level of 6.74 after 40 days of fermentation. The fermented anchovy paste attain a rather neutral pH possibly because most of the organic acids produced during fermentation is in its salt form which is comparable to the shrimp paste studied by Mizutani, et al. (1992). Comparing the

48 start and end of fermentation (D0 and D28), a slight increase in pH from 6.73 to 7.01 has been observed. This suggests that protein degradation occurs during the fermentation period of SP. However, the pH of the fermented fish at the end of fermentation is not higher than 7.0 due to its strong buffering capacity (Visessanguan & Chaikaew, 2014).

Table 15. Chemical composition of anchovy pastes in different time points of fermentation

DM Crude Fat Crude Protein NaCl T Aw pH* (g/100g) (g/100gDM) (g/100gDM) (g/100gDM) R 22±0.69a 6.44±1.47 93.1±2.04a 0.99±0.004 6.82±0.18 5.80±2.33a D0 38±1.97b 8.29±2.76 49±2.75b 0.82±0.02 6.73±0.14 30.5±11.50b D9 39±3.19b 6.11±1.06 52.8±2.54b 0.82±0.03 6.72±0.18 37.1±8.14b D19 48±9.68b 4.12±0.95 42.5±9.58b 0.82±0.03 6.94±0.19 22.2±4.48b D28 45.9±9.18b 5.24±3.71 45.2±11.65b 0.82±0.03 7.01±0.31 37.1±11.04b p-value 0.004 0.153 0.045 0.112 0.091 0.005

Results expressed as mean ± standard deviation; n=3;*-n=6 Values within the column with the different superscripts (a-e) denote significant difference from each other, p ≤ 0.05.

ii. Mineral Composition

Mineral content of SP is shown in table 16. Microelements analyzed resulted in values of 9-15, 8-26, 2-5, and 26-75 µg/g DM for Cu, Fe, Mn and Zn respectively. There were no significant differences (p˃0.05) observed between T for Cu and Mn contents. However, Fe and Zn contents varied significantly (p<0.05) between R and D0. For the macroelements, values obtained were between 1.7-3.8, 11.8-18.6, 39-48 and 23-146 mg/gDM for Mg, K, Ca and Na respectively. There were no significant differences (p˃0.05) between T for all macroelements analysed except Na where it differs significantly (p<0.05) with R and D0. In the study of Sankar, et al. (2013) on fresh anchovy, the macroelements Na and Ca are lower than SP whose values obtained were 16.57, 18.7m/gDM respectively. However, Sankar, et al. (2013) reported a higher K content in the fresh anchovy which is 54mg/gDM. Also in their study, the microelements Cu and Mn are lower than the one obtained in SP with varying values of 0.85 and 0.42mg/gDM correspondingly. It can be observed that all minerals, except Na, decreased from R to D0. The decrease is attributed to the draining process while the increase in Na is due to the addition of

49

salt (NaCl). Comparing the start and the end of fermentation, there is no significant differences in the content of the microelements. This suggests that microelements were not affected by fermentation. However, this was in contrast with the study of Bakhiet, et al., (2013) on salted Hydrocynus spp. who reported a decrease in Cu and Fe and an increase in Zn due to salting and fermentation. On the other hand, there was an increase for the macroelements from the start to the end of fermentation. The analysis was in agreement with the study of Kim, et al., (2003) where an increase of Ca, Fe and Mg was also observed from the salt-fermented shrimp sauce. Whereas in the study of Bakhiet, et al., (2013), Ca and Mg were not affected by salting and fermentation while a decrease in K is observed due to washing.

Table 16. Mineral composition of anchovy pastes in different time points of fermentation

Microelement (µg/gDM) Macroelement (mg/gDM) T Cu Fe Mn Zn Mg K Ca Na

R 14.6±7.62 26.4±7.30a 5.31 75.2±20.83a 3.81±1.31 18.6±7.48 44.1±4.09 22.8±9.16a D0 10 8.11±5.12ab 2.36 34.3±5.91b 1.71±0.74 11.8±7.75 39±18.24 119±45.21b D9 10.9±1.10 11.8±4.05ab 2.51±0.29 35.3±9.54b 2.72±0.87 14.8±5.01 51.2±17.27 145±32.01b D19 9.47±0.50 9.93±4.65b 2.14±0.08 26.5±1.63b 1.75±0.64 12.4±6.49 41.6±11.42 87.3±17.62ab D28 10.6 8.38±5.98b 2.52 31.8±5.18b 2.38±1.12 13.6±6.62 48.7±25.12 145±43.39b p-value 0.543 0.014 0.295 0.001 0.122 0.752 0.892 0.005

Results expressed as mean ± standard deviation; n=3. Values within the column with the different superscripts (a-e) denote significant difference from each other, p ≤ 0.05.

iii. Fatty Acid Composition

Total SFA, MUFA and PUFA obtained from SP are shown in table 17. Their values ranged from 38.9-49.5, 14.6-17.4, and 24-35.8g/100g FAME respectively. SFA was the predominant group of fatty acids in the sampled anchovy paste where the major SFA is C16:0. In the study of Sankar, et al. (2013) on medium-sized anchovy, SFA was also the predominating fatty acid group. Another study of fermented anchovy paste also showed SFA as the major fatty acid group (Montaño, et al., 2001). Sankar, et al., (2013) and Montaño, et al. (2001) reported that C16:0 is the major SFA which coincides with the study of El-Sebaiy & Metwalli (1989) on

50 fermented Bouri fish. The major PUFA was C22:6ω-3 which is also in agreement with the studies of Sankar, et al. (2013), Montaño, et al. (2001) and El-Sebaiy & Metwalli, (1989). There was a significant increase (p-value<0.05) in the total SFA from 39.7g/100gFAME at R to 46.5g/100gFAME at D0, while a significant decrease (p-value<0.05) was observed in the total PUFA from 34.7g/100gFAME at R to 26.3g/100gFAME at D0. On the other hand, there was no significant difference (p-value˃0.05) in total MUFA between R (14.8g/100gFAME ) and D0 (16.6g/100gFAME). The increase in total SFA from R to D0 suggests that the addition of salt increased the SFA content. This could be attributed to the halophilic bacteria inherent in the salt. Oren (2003) has stated that halophilic bacteria mainly contain common straight-chain saturated and monounsaturated fatty acids in their membrane lipids such as C16:0, C16:1 and especially C18:1. The increase in SFA is also supported by the different studies shown in table 13 where total SFA of the unfermented anchovy has a lower value than the fermented ones. On the other hand, the decrease in total PUFA from R to D0 is attributed to the decrease in C22:6ω-3, C20:5ω-3 and C 18:3ω-6. Table 13 also showed that unfermented anchovy has more PUFA than the fermented ones. The decrease is an effect of lipid oxidation, wherein PUFAs are degraded into free fatty acids, during draining as the products were exposed to air. It has been known that PUFA are highly prone to oxidation (Rashid, et al., 1992; Binsas, et al., 2008). Comparing the start and end of fermentation, there was no significant difference in the total SFA, MUFA and PUFA. This suggests that fatty acids are not affected by fermentation which is also in consistent to the one reported by Montaño, et al. (2001) on fermented shrimp paste. Looking at the individual PUFA, C22:6ω-3 (DHA) has the highest value. This indicate that the fermented anchovy paste is rich in DHA which is essential for the normal functioning and development of retina and brain of humans, particularly in infants (Sankar, et al., 2013).

Total ω3 of SP obtained in this study varied between 21.4-29.4g/100g FAME in which R (29.4g/100gFAME) significantly decrease (p-value <0.05) to D0 (21.4g/100gFAME). This decrease is attributed to the decrease in its individual ω3, C20:5ω-3 and C22:6ω-3. This decrease is also supported by the studies of Sankar, et al. (2013), Montaño, et al. (2001) and belacan and kapi from Sumino, et al. (1999,2003) in table 13. Their studies showed a lower total ω3 value in the fermented anchovy pastes than the unfermented anchovy. Comparing D0 to D28, no significant differences (p-value˃0.05) were observed. Total ω6 of SP varies from 4.8-

51

5.36g/100gFAME and the results showed no significant differences (p-value˃0.05) from the different T.

Table 17. Fatty acid profile of anchovy pastes in different stages of fermentation

FAME Time point (T) p-value (g/100g) R D0 D9 D19 D28 C 12:0 0.14±0.03 0.17±0.01 0.18±0.007 0.17±0.03 0.17±0.04 0.536 C 13:0 0.12±0.02 0.12±0.04 0.11±0.03 0.09±0.03 0.15±0.02 0.210 C 14:0 3.83±0.17 4.14±0.51 4.46±0.22 4.28±0.17 4.67±0.59 0.149 C 15:0 1.62±0.10 1.82±0.20 1.73±0.23 1.62±0.13 1.89±0.23 0.349 C 16:0 22.2±1.47a 26.2±1.06b 24.9±0.44ab 25.9±1.22b 25.1±1.6ab 0.018 C 17:0 2.22±0.17 2.50±0.13 2.41±0.24 2.34±0.07 2.55±0.14 0.167 C 18:0 9.16±0.89 11±1.09 10.5±0.89 10.3±0.94 11.4±0.60 0.089 C 20:0 0.23±0.02 0.29±0.005 0.28±0.05 0.35±0.06 0.30±0.05 0.067 C21:0 0.06±0.03 0.09±0.01 0.10±0.03 0.12±0.04 0.10±0.06 0.472 C22:0 0.17±0.05 0.23±0.02 0.25±0.03 0.23±0.06 0.23±0.05 0.353

C 14:1 0.25±0.11 0.29±0.10 0.37±0.19 0.33±0.06 0.33±0.16 0.845 C 15:1 0.30±0.17 0.34±0.17 0.47±0.07 0.46±0.15 0.39±0.05 0.489 C 16:1 4.35±0.48 4.70±0.53 4.57±0.66 4.51±0.16 5.02±0.31 0.509 C 17:1 1.26±0.16 1.33±0.09 1.24±0.08 1.26±0.20 1.37±0.06 0.697 C 18:1 7.98±0.78 9.48±1.15 8.72±1.06 9.45±0.73 8.61±0.51 0.259 C20:1 0.73±0.24 0.49±0.11 0.52±0.06 0.58±0.08 0.69±0.10 0.179

C 18:2ω-6 1.59±0.20 1.67±0.11 1.62±0.04 1.60±0.02 1.72±0.20 0.735 C 18:3ω-6 0.53±0.16a 0.45±0.12b 0.45±0.07ab 0.72±0.24ab 0.45±0.11ab 0.039 C20:2ω-6 0.19±0.03 0.29±0.10 0.24±0.02 0.21±0.02 0.21±0.02 0.277 C20:4ω-6 3.05±0.33 2.45±0.11 2.49±0.14 2.82±0.25 2.51±0.34 0.062

C18:3ω-3 0.86±0.23 0.74±0.17 0.76±0.13 0.75±0.14 0.94±0.08 0.507 C20:5ω-3 4.55±0.19a 3.33±0.22b 3.69±0.13ab 4.0±0.30ab 3.65±0.72ab 0.025 C22:5ω-3 1.81±0.44 1.80±0.72 1.63±0.70 1.84±0.29 2.13±0.70 0.885 C22:6ω-3 22.1±1.29a 15.6±1.77b 17.9±0.87ab 18.4±1.07ab 16.8±4.16ab 0.039

Total SFA 39.7±2.97a 46.5±3.07b 44.9±2.16b 45.4±2.76b 46.6±3.36b 0.003 Total MUFA 14.9±1.93 16.6±2.14 15.9±2.13 16.6±1.37 16.4±1.18 0.068 Total PUFA 34.7±2.87a 26.3±3.31b 28.8±2.08ab 30.3±2.33ab 28.4±6.34ab 0.018 Total ω3 29.4±2.15a 21.4±2.88b 24±1.82ab 24.9±1.80ab 23.5±5.66ab 0.024 Total ω6 5.36±0.72 4.86±0.44 4.8±0.27 5.35±0.53 4.89±0.67 0.644 Results expressed as mean ± standard deviation; n=3. Values within the row with the different superscripts (a-e) denote significant difference from each other, p ≤ 0.05.

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iv. Non-protein and TBARS composition

Total and free NPN content of SP are shown in table 18. Total NPN varied between 54- 386 mg/100gDM where there was a significant decrease (p<0.05) from R to D0. Total NPN increases from D0 to D9and then rest stable. Free NPN content varied between 9- 38mg/100gDM. The analysis showed significant differences (p<0.05) between T. Free NPN content decreased from R to D0 and then began to increase until the end of fermentation. The decrease in NPN (total and free) can be due to the removal of some NPN as a result of the draining at D0. On the other hand, the increase in total and free NPN from the start to the end of fermentation is the result of protein hydrolysis (Ng, et al., 2011; Kilinc, et al., 2005).

Values of TBARS analyzed in SP ranged between 3-18µgMDA/gDM. There was an increase in TBARS content when salt was added at D0 then follows a decreasing trend from D9 to D28. The increase is due to the exposure of lipids to air when they were drained. Then as fermentation goes on, TBARS decreases due to anaerobic condition restricting the reaction of oxygen and lipids. In the study of Montaño, et al. (2001) on fermented shrimp paste, it was said that oxidation was significantly suppressed during fermentation. Peralta, et al. (2008) also observed an increase in antioxidant activity in the fermented shrimp paste causing PUFAs to remain intact even at longer fermentation period thus not much of it is involved in lipid oxidation. Table 18. Non-protein nitrogen and TBARS of anchovy paste traditionally produced in some parts of the Philippines

Time point Free NPN Total NPN TBARS (mg/100gDM) (mg/100gDM) (µgMDA/gDM) R 31.9±4.05a 234±94.37a 9.90±2.96a

D0 10.1±0.90b 88.9±35.18b 14.7±3.70ab

D9 22±5.05c 125±50.64b 8.65±2.99ac

D19 25.4±5.09ac 116±17.25b 4.70±2.09c

D28 30.3±5.04ad 116±13.95b 4.12±2.03c p-value < 0.001 0.006 < 0.001

Results expressed as mean ± standard deviation; n=6. Values within the column with the different superscripts (a-e) denote significant difference from each other, p ≤ 0.05.

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b. Microbial Characterization

The liquid part of the fermented anchovy pastes with different T were also collected and analysed for microbial characterization. The total Enterobacteriaceae, aerobic bacteria, halophilic aerobic bateria, lactic acid bacteria (LAB), halophilic LAB, proteolytic bacteria and halophilic yeasts were analysed.

The analysis showed no counts of Enterobacteriaceae. Enterobacteriaceae are gram- negative, non-spore-forming bacteria of which some of them are important human and animal pathogens such as Escherichia, Shigella, Salmonella and Yersinia. They cause foodborne diseases as well as food spoilage. They are used as an indicator organism for poor hygiene, inadequate processing or post-process contamination of foods (Baylis et al., 2011). With this result, it suggests that fermentation process was done hygienically.

1.00E+08

1.00E+07

1.00E+06

1.00E+05

1.00E+04

1.00E+03

Microbial Count (CFU/mL) Count Microbial 1.00E+02

1.00E+01

1.00E+00 D0 D9 D19 D28 Fermentation Day

LAB halophilic LAB halophilic yeast

total proteolytic total aerobic total halophilic aerobic

detection limit

*points on the detection limit line are below the detection limit* Figure 4. Microbial Count of fermented anchovy pastes on different days of fermentation

54

Figure 4 showed the growth of the different microorganisms involved during fermentation of SP. The total aerobic count increased up to 106CFU/mL from D0 to D19 and then slightly decreased to 105CFU/mL with D28. In the case of total halophilic bacteria, they were below the detection limit at the start of fermentation. But they gradually increased to 106CFU/mL at D19 and then decreased slightly to 105CFU/mL with D28. Chinte-Sanchez (2008) has cited similar results obtained from nampla where the total bacterial count increased up to 108 CFU/mL on the third week of fermentation, and then a slow decrease after six months was observed. Also during the first 10 days of fermentation in the study on Indonesian fermented fish sauce by Ijong & Ohta (1996), total bacterial count increased significantly then gradually decreased thereafter. According to Jiang et al. (2007), there is a high bacterial count in the early stage of fermentation because salt has not yet completely dissolved and penetrated into the fish flesh. After several months, non-halophilic bacteria disappeared while halotolerant and halophilic bacteria remain. These halotolerant and halophiles played a main role in fermentation.

Lactic acid bacteria (LAB) and halophilic LAB were already present even from the start of the fermentation. Their count both increase from 103 to 106CFU/mL and 104 to 107CFU/mL respectively until D19 but then both decreased to 105CFU/mL at D28. It was the halophilic LAB which has a higher count than LAB. This shows that halophilic LAB are the dominant microorganisms involved during the fermentation process. In the study of Ijong & Ohta (1996) on Indonesian fermented fish sauce, LAB increased during the first 10 days of fermentation then gradually decreased thereafter. A similar result was also obtained in the study of Kilinc, et al. (2005) on fish sauce where LAB increased until day 8 of fermentation corresponding to a decrease in pH. Afterwards, LAB count decreased. LAB are mesophilic but some species are able to grow at low temperature (as low as 5°C) and high temperature (as high as 45°C) (Sivasankar, 2005). LAB produces lactic acid from carbohydrates lowering the pH of food. They also produce bacteriocins inhibiting growth of pathogens and spoilage microorganisms. Moreover, they modify the raw material to obtain new sensory properties as well as improving the shelf-life of fish products (Thienchai & Chaiyanan, 2012). LAB also contributes to the flavor development of fish sauces (Kilinc, et al., 2005). Leuconostoc, Lactobacillus, Streptococcus and Pediococcus are among the species which belong to the group of LAB. The increase in the

55 number of LAB should correspond to the decrease in pH due to the production of lactic acid. However, in this study, there is a minimal change in the pH. Though there is an increase of LAB in the sample, the lactic acid produced by these microorganisms may not be enough to decrease the pH compared to the alkalinity caused by proteolysis of the fish samples. This is due to the buffering capacity of the fermented fish and organic acids produced are in salts form (Visessanguan & Chaikaew, 2014; Mizutani, et al., 1992).

For proteolytic bacteria and halophilic yeasts, they were not yet detected at the start of fermentation but both started to increase up to 106CFU/mL on D19, and then a gradual decrease was observed to 104CFU/mL on D28. Proteolytic bacteria are bacteria which produces protease that split proteins into peptides and amino acids. By the breakdown of proteins, the structures of food products are changed (Sivasankar, 2005; Brown, 2011;). Species of these bacteria include aerobic, facultative and spore-forming organisms. Clostridium, Bacillus, Pseudomonas and Proteus are the dominant species belonging to this group (Sivasankar, 2005). It was observed in this study that at D19, where the count of proteolytic bacteria was at its highest, the pH started to increase slightly. Higher pH allows the bacteria to become dominant and also favors the breakdown of proteins that releases amine compounds (Visessanguan & Chaikaew, 2014).

Yeasts help in the synthesis of essential amino acids such as glutamic acid and lysine which enhances the flavor of fish paste (Chinte-Sanchez, 2008). Yeasts were also identified by Thapa,et al. (2004) in their study on ngari, a fermented fish product. The yeasts involved were Candida and Saccharomycopsis. In the study of Sanni, et al. (2002) on fermented fish momoni, they have identified the yeasts Debaryomyces hansenii and Hansenula anomala. Likewise, Crisan and Sands (1975) reported Candida clausenii to be present in patis.

It can be observed that numerous microorganisms identified in the study are halophilic bacteria. Halophilic bacteria require a certain minimal salt (NaCl) concentration for their growth. They can survive even at high salt concentration depending on the type of the species which could be slightly, moderately or extremely halophilic or halotolerant. Species belonging to this type of bacteria include Bacillus, Micrococcus, Vibrio, Moraxella, Halobacterium, Corynebacterium, Streptococcus and Clostridium (Sivasankar, 2005). According to Chinte-

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Sanchez (2008), the microorganisms which have a main role in fermentation of fish paste were the halophilic bacteria. These bacteria increased rapidly because of the favorable growth condition from the nutrients in brine. She also described that Bacillus, and not halophilic LAB,where the predominant bacteria throughout the fermentation suggesting that Bacillus have an active role in the process. This is also supported by Sanni (2002) stating that Bacillus were the predominant bacterial flora in the fermented fish. In the study of Sarojnalini & Suchitra (2009, 2012), Bacillus and Micrococcus were the predominant microorganisms involved in the fermentation of ngari. These bacteria have proteolytic and lipolytic activity which contribute to the development of the typical odour and flavour of the final product (Sarojnalini & Suchitra, 2009,2012). Enzymes from Bacillus subtilis and B. coagulans have minimal role in proteolysis during the early stage but are mainly responsible for the production of flavors (Chinte-Sanchez, 2008). Different authors enumerated halophilic bacteria like Bacillus, Micrococcus, Pediococcus, Moraxella, LAB and some mould and yeasts dominate in the ripening of fermented fish products. They are important for fish tissue breakdown and generation of aroma and flavour (Majumdar & Basu, 2010; Aryanta, 2000).

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Chapter 5. Conclusions and Recommendation

The anchovy paste products in the Philippines are microbiologically stable. Due to its low

Aw and high salt content, spoilage microorganisms are inhibited. The product is predominant in SFA and C16:0 is the major SFA. It is also rich in PUFA, particularly in DHA which is essential for infant’s brain development. Moreover, it is also rich in proteins and minerals (Na, Ca, K, Zn, Fe) which are essential to human diet.

Gross composition of the anchovy pastes from the different producers didn’t vary significantly. Though they were processed in different techniques, they still conform to the standards for bagoong set by the Philippine Standard Association. However, there is significant difference in the pH, fatty acid, NPN and TBARS to which the cause might be attributed to the ripening period.

The amount and level of proteins, Aw, pH, minerals, PUFA, ω3, free and total nitrogen of the raw anchovy decrease at the start of fermentation period. On the other hand, an opposite is observed for DM, fats, salt, SFA and TBARS content. The difference is caused by the draining, addition of salt and exposure of the product to air during draining.

Fermentation increases the concentration of DM, salt content, total and free NPN of the salt-fermented anchovy paste. This is due to the effect of osmosis and proteolysis. In contrast, fermentation decreases the content of fats, proteins and TBARS which is caused by fat degradation, protein hydrolysis and restriction of aerobic condition. Fermentation does not affect significantly the Aw, mineral content and fatty acids of the salt-fermented anchovy paste.

The microbial count of the fermented anchovy paste increases and is at its highest on D19 then decrease gradually on D28. Enterobacteriaceae are not detected in the studied fermented anchovy pastes which show that the product is processed hygienically. Aerobic bacteria, LAB and halophilic LAB are involved at the start of fermentation. Proteolytic, halophilic aerobic bacteria and halophilic yeasts play a role starting at the middle period of fermentation. The fermentation of the anchovy pastes is predominated by halophilic bacteria. Halophilic LAB

58 attain the highest count however, the pH of the fermented product doesn’t decrease that much because most of the acids produced where in its salt form. The fermented fish also has a neutral pH rather than higher pH because of the buffering capacity of the fermented fish. Based on the values of pH obtained and literatures gathered, it can be concluded that it is Bacillus species that play a vital role in the fermentation process.

Further studies regarding the salt-fermented anchovy pastes particularly on the identification of the species of halophilic bacteria involved in the fermentation process are deemed necessary. Also, a study should be taken on the characterization of the amino acids and biogenic amines of the salt-fermented anchovy pastes during the different stages of fermentation as these were not analyzed by the present study. Since the samples were stored for a year already before analyzed, it would be interesting to conduct a similar study with samples gathered earlier to compare and verify the results obtained in this study. It would also be interesting to conduct a similar research on salt-fermented anchovy pastes from the other areas of the Philippines.

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