FACULTY OF BIOSCIENCE ENGINEERING INTERUNIVERSITY...
Transcript of FACULTY OF BIOSCIENCE ENGINEERING INTERUNIVERSITY...
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 ANCHOVY PASTE 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
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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 anchovy paste or
bagoong 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. Fermented Fish Products in Thailand ......................................................................................... 7
D. Fermented Fish Products in Indonesia ....................................................................................... 8
E. Fermented Fish Products in Myanmar ...................................................................................... 11
F. Fermented Fish Products in Malaysia ....................................................................................... 11
G. Fermented fish products in Vietnam ........................................................................................ 14
H. Fermented Fish Products in Philippines ................................................................................... 15
I. Fermented Fish Paste 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 Sauce 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) lactic acid fermentation; 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 cooking 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 fish preservation 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 fish sauce. They have salty, slightly cheese-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, ngapi 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,
anchovies, 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
salad dressings. It is also used as ingredients in different sauces (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
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intoxication that would result in food safety issues (Chojnacka, 2010). Fermented foods can be
eaten cooked or uncooked and even used as condiments (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, shellfish, and crustaceans which are processed with salt to
undergo fermentation thus, preventing spoilage (Ishige, 1993). Fermented fish differs from salted
fish 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 rice, 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/Shrimp Paste Fish/Shrimp Sauce
China
Yu-lu
Cambodia Prahoc Nuoc-mam
Indonesia Trassi; Trassi-ikan; trassi udang Ketjap-kan; Kecap-ikan
Japan Shiokara Shottsuru
Korea Jeotkal Jeot-kuk
Laos Pradec Padec
Malaysia Belachan Budu; 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
mussels 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
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microorganisms involved in the fermentation, such as lactic acid bacteria, and for taste
development of the product. Products with cooked rice 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 glutinous rice (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
catfish) 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),
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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, garlic 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
10
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 soy sauce. 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
Components Pedaha
Shrimp
Pasteb
Fish
Saucec
Bekasamd Jambal roti
e
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
sample) 52.12 20-40 10-10.5 41-64 54.17-61.86
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
11
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
12
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 condiment. 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, tamarind, 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
13
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 Pekasam
b Belacan
c
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
14
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 syrup
(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
15
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 (burong isda),
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
16
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, milkfish, Ophicephalus striatus, Tilapia 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).
17
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
18
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
3.6x10-1
cfu/g and 9x10-1
cfu/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
19
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
20
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
21
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:
𝐷𝑀 = (𝑀2 − 𝑀0)
(𝑀1 − 𝑀0) 𝑥 100
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
22
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:
𝐸 = ((𝑉 − 𝑉𝑏) ∗ 14 ∗ 𝑁 ∗ 6.25
𝑉𝑠 ∗ 𝐷𝑀 ∗ 1000) ∗ 100
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)
23
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:
𝑉 = [(𝐾2 − 𝐾1
𝑀1 − 𝑀0) 𝐷𝑀⁄ ] 𝑥 100
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)
24
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.
25
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:
𝐶𝑜𝑛𝑐𝑚𝑎𝑐𝑟𝑜 =(𝑐 ∗ 𝑑)/1000
𝑉𝑠 ∗ 𝐷𝑀
𝐶𝑜𝑛𝑐𝑚𝑖𝑐𝑟𝑜 =(𝑐 ∗ 𝑑) ∗ 1000/1000
𝑉𝑠 ∗ 𝐷𝑀
Where:
Concmacro = concentration of each macroelement (mg/g DM)
26
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:
𝑁𝑎𝐶𝑙 = 𝑎 𝑥 𝑏
𝑐
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
27
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)
28
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:
𝑃𝑟𝑜𝑝 = (𝑋
𝑌) ∗ 100
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)
29
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.
30
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:
𝑇𝑜𝑡𝑎𝑙𝑁𝑃𝑁 =𝑥∗𝑑∗50∗ 100∗100
𝑉𝑠∗𝐷𝑀∗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
31
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:
𝐹𝑟𝑒𝑒𝑁𝑃𝑁 =𝑥∗𝑑∗100∗100
𝑉𝑠∗𝐷𝑀∗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)]
32
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:
𝑇𝐵𝐴𝑅𝑆 = 𝑐 𝑥 20 𝑥 𝑑 𝑥 72
𝑚 𝑥 𝐷𝑀∗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)
33
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
34
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:
𝑋 = 𝐴 𝑥 𝑉
𝑙
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.
35
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
Name of Manufacturer Date/Time
Manufactured Region of Origin
Other
Characteristics
SPA. Gimaylan Salted Fish
Processors Organization (6 years experience)
July 2013
8AM
Misamis Oriental
1:3.6 salt:fish
ratio
fish used was
caught on the previous day and
stored at -10°C
before processing
unwashed fish
liquid drained
SPB. Gimaylan Salted Fish Processors Organization
(6 years experience)
July 2013
10AM
1:3.6 salt:fish
ratio
used freshly
caught fish from local fishermen
unwashed fish
liquid drained
SPC. Gimaylan Salted Fish Processors Organization
(6 years experience)
July 2013
1PM
1:3.6 salt:fish
ratio
fish used from earlier catch of
local fishermen
unwashed fish
liquid drained
FPA. Escalona (20 years experience)
May 2013 Surigao del Norte
salt and fish ratio
not specified
FPB. Millan
(5 years experience) July 2013 Surigao del Sur
FPC. Durando (17 years experience)
April 2013 Surigao del Sur
36
Figure 2. Fermented anchovy pastes at different time points
Figure 3. Fermented anchovy pastes locally sold in markets
37
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).
38
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±0.02
b 6.55±0.14
c 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
39
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
40
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
Mineral Sample
FPA FPB FPC p-value
Microelement
(μg/g DM)
Cu ND ND ND
Fe 18.9±6.25 ND 15.8±5.14 0.643
Mn ND ND ND
Zn 5.39±1.71a 28.1±0.39
b 20.9±6.01
b 0.018
Macroelement
(mg/g DM)
Mg 1.25±0.003a 1.95±0.18
b 11±1.38
c <0.001
K 15.9±10.03 14.9±2.0 12.5±1.56 0.215
Ca 36.4±1.69a 49.5±6.91
ab 67.7±8.58
b 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
41
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
42
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.
43
Table 12. Fatty acid profile (g/100g FAME) of anchovy paste traditionally produced in some
parts of the Philippines
FAME (g/100g) Sample
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±0.13
b 5.38±0.34
ac 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±0.26
ab 23.5±1.04
ac 0.022
C 17:0 2.42±0.08a 2.63±0.009
ab 2.26±0.12
ac 0.046
C 18:0 8.35±0.07a 12.94±0.3
b 10.1±0.26
c 0.001
C 20:0 0.31±0.004a 0.23±0.002
b 0.25±0.02
b 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±0.005
ab 0.54±0.04
c 0.005
C 16:1 4.48±0.14a 7.13±0.45
b 3.82±0.22
ac 0.003
C 17:1 0.85±0.09a 1.28±0.02
b 0.88±0.02
ac 0.013
C 18:1 8.1±0.18a 8.8±0.05
ab 7.68±0.31
ac 0.028
C20:1 1.1±0.02a 0.49±0.02
b 0.72±0.07
c 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±0.07
b 0.85±0.04
ab 0.045
C20:5ω-3 6.09±0.06a 2.77±0.16
b 4.38±0.35
c 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±0.89
b 16.7±0.61
ac 0.002
Total SFA 44.8±1.00a 53.4±0.98
b 44.5±2.2
ac 0.012
Total MUFA 15±0.46a 18.4±0.74
b 14±0.69
ac 0.009
Total PUFA 31.3±0.47a 18.9±1.73
b 28.1±1.92
ac 0.003
Total ω3 26.9±0.28a 14.6±1.46
b 23.6±0.63
ac 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.
44
Table 13. Fatty acid profile of anchovy pastes obtained from different studies
FAME (g/100g) Montaño
(2001)
Sankar, et al.,
(2013)*
Belacan (Sumino,
et al., 2003)
Kapi (Sumino,
et al., 1999)
Bagoong
(Sumino, et al.,
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
45
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.
46
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
Composition Sample
A B C p-value
Free NPN (mg/100gDM) 20.2±0.25a 12.4±4.04
b 16.9±0.74
b 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±1.95
ab 13.2±3.26
c 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.
47
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
48
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
49
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
T DM
(g/100g)
Crude Fat
(g/100gDM)
Crude Protein
(g/100gDM) Aw pH
*
NaCl
(g/100gDM)
R 22±0.69a 6.44±1.47 93.1±2.04
a 0.99±0.004 6.82±0.18 5.80±2.33
a
D0 38±1.97b 8.29±2.76 49±2.75
b 0.82±0.02 6.73±0.14 30.5±11.50
b
D9 39±3.19b 6.11±1.06 52.8±2.54
b 0.82±0.03 6.72±0.18 37.1±8.14
b
D19 48±9.68b 4.12±0.95 42.5±9.58
b 0.82±0.03 6.94±0.19 22.2±4.48
b
D28 45.9±9.18b 5.24±3.71 45.2±11.65
b 0.82±0.03 7.01±0.31 37.1±11.04
b
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
50
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
T Microelement (µg/gDM) Macroelement (mg/gDM)
Cu Fe Mn Zn Mg K Ca Na
R 14.6±7.62 26.4±7.30a 5.31 75.2±20.83
a 3.81±1.31 18.6±7.48 44.1±4.09 22.8±9.16
a
D0 10 8.11±5.12ab
2.36 34.3±5.91b 1.71±0.74 11.8±7.75 39±18.24 119±45.21
b
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±32.01
b
D19 9.47±0.50 9.93±4.65b 2.14±0.08 26.5±1.63
b 1.75±0.64 12.4±6.49 41.6±11.42 87.3±17.62
ab
D28 10.6 8.38±5.98b 2.52 31.8±5.18
b 2.38±1.12 13.6±6.62 48.7±25.12 145±43.39
b
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
51
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-
52
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
(g/100g)
Time point (T) p-value
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±1.06
b 24.9±0.44
ab 25.9±1.22
b 25.1±1.6
ab 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±0.12
b 0.45±0.07
ab 0.72±0.24
ab 0.45±0.11
ab 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±0.22
b 3.69±0.13
ab 4.0±0.30
ab 3.65±0.72
ab 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±1.77
b 17.9±0.87
ab 18.4±1.07
ab 16.8±4.16
ab 0.039
Total SFA 39.7±2.97a 46.5±3.07
b 44.9±2.16
b 45.4±2.76
b 46.6±3.36
b 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±3.31
b 28.8±2.08
ab 30.3±2.33
ab 28.4±6.34
ab 0.018
Total ω3 29.4±2.15a 21.4±2.88
b 24±1.82
ab 24.9±1.80
ab 23.5±5.66
ab 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.
53
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
(mg/100gDM) Total NPN
(mg/100gDM)
TBARS
(µgMDA/gDM)
R 31.9±4.05a 234±94.37
a 9.90±2.96
a
D0 10.1±0.90b 88.9±35.18
b 14.7±3.70
ab
D9 22±5.05c 125±50.64
b 8.65±2.99
ac
D19 25.4±5.09ac
116±17.25b 4.70±2.09
c
D28 30.3±5.04ad
116±13.95b 4.12±2.03
c
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.
54
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.
*points on the detection limit line are below the detection limit*
Figure 4. Microbial Count of fermented anchovy pastes on different days of fermentation
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
D0 D9 D19 D28
Mic
rob
ial C
ou
nt
(CFU
/mL)
Fermentation Day
LAB halophilic LAB halophilic yeast
total proteolytic total aerobic total halophilic aerobic
detection limit
55
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 10
5CFU/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 10
6CFU/mL and 10
4 to 10
7CFU/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
56
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-
57
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).
58
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
59
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.
60
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