Chapter 2

Literature Review

Fermentation is a method of preservation of food by using microorganisms that have

been utilized by humans for thousands of years. The food or its products where the
controlled microbial growth performs the biotransformation into new edible forms
with enhanced flavors and aromas are called fermented foods (Xiang et al., 2019).
The microbial population in fermented food products causes chemical and textural
changes (Kavitake et al., 2018). According to Steinkraus (1993), fermented food is a
fascinating relationship between man, microbes, and food. The microbial population
in fermented food can cause spoilage or produce a food product acceptable to man.
So, there is a very thin line between rotten and fermented food, which is demarked by
taste and tradition (Steinkraus, 2004). The first one is undesirable, whereas the latter
is considered a delicacy. Fermented food helped to sustain the human population in
the past and provided proteins, vitamins, minerals, and calories. The co-evolution of
food and human civilization is a known fact, and so is the fermentation technique
adopted to preserve perishable food items (Anal et al., 2019).

2.1 History of fermented food

Alcohols were the first fermented product consumed by man, and its scanty evidence
dates back at least 30,000 years ago. The first Neolithic jars consisting of a chemically
fermented mixture discovered in Jiahu (Hunan Province, China) date back to 6500-
5500 B.C (McGovern et al., 2004). Dominy, 2015 stated that alcoholic fermentation is
a natural process where primates, birds, and insects also partake in its consumption
unintentionally. He opined that human beings and the food habit consisting of
fermented food co-evolved in due process of human civilization. Kindstedt (2012) in
his book “Cheese and Culture: A History of Cheese and Its Place in Western
Civilization” reported that fermented product known in the Neolithic age was
fermented milk products like curd and cheese, and the art of cheese-making was
developed as far back as 8000 years ago in the Fertile Crescent between Tigris and
Euphrates Rivers in Iraq. The Egyptians developed dough fermentations used in
leavened bread in 4000–3500 BCE (Prajapati & Nair, 2003). The Harappans (3200 –
1500 BC) appeared to have known the process of alcoholic fermentation and its
distillation (Mahdihassan, 1979). Mahdi Hassan reported that even before the advent

6
of Aryans, the earliest distilled liquor in India was Parisrut prepared from the flowers
of Mahua (Bassia latifolia) which dates back to 3000 B.C. (Mahdihassan, 1981).
Alcoholic beverages are still known as Mahua in Bihar. In the Vedic age, there is
ample evidence of the use of fermentation technology in 1500 BC-600 BC as the
Rigveda mentions the names of three fermented beverages somrasa, sura,
and parisrut (Chaudhary, 2019).

2.2 Types of fermented food

There are several types of fermented food products and beverages consumed
worldwide (viz: kefir, sauerkrant, miso, salami, cheese, idli, dosa, tempe, natto, beer
wines, pickles, etc.). Tamang (2010) classified fermented foods based on the pattern
of fermentation, use of micro-organisms, and substrate in fermentation. 1) Based on
the pattern of fermentation, they grouped the products into solid
(e.g., Tempe), submerged (e.g., soy sauce), and moist types. 2) Based on raw materials
or substrates, there are eight major groups, viz. fermented vegetable, fermented
soybean, and non-soybean legumes, fermented cereals, fermented milk, fermented
fish, fermented roots, and tubers, and miscellaneous. 3) Based on the micro-organism,
fermented food may be naturally fermented or by the addition of external ‘starter
culture’. Most fermented food products are acidic, but some are alkaline (Wang &
Fung, 1996). Alkaline-fermented foods constitute a group of food products widely
consumed in Southeast Asia and African countries. In alkaline fermented food
products, as the name suggests, the pH tends to increase during fermentation (Nout,
2014). Most alkaline fermentations are achieved spontaneously by mixed bacterial
cultures, principally dominated by Bacillus subtilis (Wang & Fung, 1996). Some
examples of Alkaline fermented foods are natto, thua-nao, and kinema prepared by
fermenting cooked soybeans; dawadawa from African locust beans; ogiri from melon
seeds; ugba from African oil beans; kawal from fresh legale leaves; owoh from cotton
seeds; and pidan from fresh poultry eggs (Sumi et al., 1987; Chukeatirote, 2015;
Sarkar et al.,1994; CampbellPlatt, 1980; Odunfa,1981; Njoku & Okemadu 1989;
Dirar, 1984; Wang & Fung, 1996; Ganasen & Benjakul, 2010).

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2.3 Health benefits of fermented food

Steinkraus (2004) enumerated the several benefits of fermented food, which included
enrichment of diet through the development of various flavors, aromas, and texture;
preservation of food for longer shelf life, enrichment of food biologically with
essential nutrients; detoxification of undesirable substances in food; reduction of
cooking time and thereby saving the fuel. Fermented foods have been a source of
supplements of essential amino acids, especially in developing countries. It can
supply beneficial nutrients to undernourished obsessed people of developed and
wealthy nations. In the past, the health benefits of fermented foods were not well
studied, so people primarily used fermentation to preserve foods, enhance shelf life,
and improve flavour. Antimicrobial metabolites such as bacteriocin are produced in
fermented food by the beneficial bacterial population that inhibits foodborne
pathogens (Kormin, 2001). Many organoleptic properties, vitamins, and minerals are
present in fermented food products (Hwang et al., 2017). The probiotics in fermented
food battle cardiovascular diseases, and gastrointestinal tract disorders, enhance the
bioavailability of nutrients, improve the immune system, and fight cancer (Ishimwe et
al., 2015). Some of the health benefits of fermented food reckoned by Farhad et al.
(2010) are reduction of blood cholesterol, increase in immunity, fight against
pathogens, fighting carcinogens, osteoporosis, diabetes, obesity, allergies, and
atherosclerosis, and alleviating the symptoms of lactose intolerance. The health
benefit of fermented is due to the bioactive peptides synthesized by micro-organisms
during biotransformation in fermented foods (Bhat, 2015). Bioactive peptides are
released during proteolysis or fermentation and play a significant role in human health
by affecting the digestive, endocrine, cardiovascular, immune, and nervous systems
(Bhat, 2015). Studies in the field of bioactive peptides have shown that dietary
proteins could be a natural source of alpha-glucosidase and DPP-IV inhibitory
peptides (Patil et al., 2015). In type 2 diabetes (T2D), Alpha-glucosidase and
dipeptidyl peptidase IV (DPP-IV) enzymes play a significant role in its development.
Hence, reduction or inhibition of their activity can be one of the crucial strategies in
T2D management. Lule et al. (2015) reported that soyderived bioactive peptide,
lunasin, possesses antioxidative, antiinflammatory, and anticancerous properties that
could play a vital role in regulating cholesterol biosynthesis in the body. Due to the
known antihypertensive effects of angiotensin-1-converting enzyme (ACE) inhibitor

8
peptides, especially valyl-polyproline (VPP) and isoleucyl-prolyl-proline (IPP),
fermented dairy items are the non-pharmacological system for the administration of
hypertension (Beltran-Barrientos et al., 2016; Nejati et al., 2013; Usinger et al. 2012).
Exopolysaccharides produced by certain strains of lactic acid bacteria are known for
their anti-oxidant, anti-diabetic, anti-carcinogenesis, cholesterol-lowering, and
immunomodulatory properties. (Pan & Mei, 2010; Wang et al., 2010; Zhang et al.,
2013). Lactic acid bacteria (LAB) gained the status of GRAS (generally recognized as
safe) because of the production of EPS, which has immense commercial importance
due to the beneficial physicochemical properties they exhibit (Surayot et al., 2014).
The bacterial exopolysaccharides can reduce total serum cholesterol by binding
cholesterol, reducing cholesterol absorption, and inducing the release of bile acids
similarly as executed by the dietary fiber (Mumford et al., 2010; Nampoothiri et al.,
2017; Tok and Aslim 2010; ùanlier, 2019). In addition to reducing cholesterol,
exopolysaccharides play a crucial role in host-microbial interactions. They are
involved in microbial colonization, attachment, and immunomodulation, and they
protect the bacterial wall against extreme conditions such as temperature fluctuations,
osmotic stress, pH changes, or light intensity which helps bacteria to survive in
extreme conditions (Caggianiello et al., 2016; Fanning et al., 2012; ùanlier, 2019).

2.4 Fermented Fish

Fish is an excellent source of low-fat proteins, omega-3 fatty acids, vitamin D,

Riboflavin, essential amino acids, and minerals (Mendivil, 2020). However, the high
moisture content and richness in nutrients increase the perishability of fish, and
processing and preservation are mandatory for fish soon after their harvesting.
Various methods like salting, drying, pickling, smoking, freezing, chemical treatment,
fermentation, and sometimes a combination of all are used for the preservation of fish
(Kolawole et al., 2010). In ancient times drying, salting and fermentation were the
traditional methods for preserving fish (Skåra et al., 2015). These traditional methods
of fish processing, collectively known as curing, were the best and cheapest method
of fish preservation in the absence of modern techniques in the past, which gradually
became part of the regular diet in many countries due to their characteristic texture
and flavor (Kumar & Nayak, 2015). Across many countries and traditions, people
developed a means to preserve food resources like fish whenever there was a seasonal
scarcity, preceded by an abundant supply of fish (Katz, 2012). The earliest fermented

9
fish sauce recorded was garum from the ancient Roman era (Curtis, 1991). In ancient
Jordan, Haimation was a highly prized fish sauce. It was previously documented only
in the ancient text, but the tracing of fish remains was reported in 2008 in an early
Roman ceramic jar from Aila Aqaba, Jordan (Van Neer, 2008). In East and Southeast
Asian countries like Korea, Japan, China, Thailand, Kampuchea, Malaysia, the
Philippines, and Indonesia, the use of fermented fish has been of great cultural value
from the distant past (Prajapati & Nair, 2003). In Northern Europe, and Africa
fermented fish products are used mainly as condiments. In India, it is an integral part
of food culture in the Northeast and Southern regions (Singh, 2014; Beddows, 1998).

2.5 Origin and cultural perspective of fermented fish

The origin of fermented fish took place independently in different locations of the
world (Ishige, 1993). (i) The center of salt production; (ii) ecological distribution of
irrigated rice cultivation, and; (iii) seasonal behavior of fish stocks in the locality were
the three criteria that led to the origin of the fermented fish product (Ishige, 1993).
Ruddle and Ishige (2010) tried to trace the likely origin and courses of distribution,
together with the development of fermented fish culture in South East Asia. From the
extensive field survey of the various fermented fishery products in countries of
Southeast Asian countries, they concluded that the widest varieties of fermented fish
products were found in Southeast Asia (Rudle & Ishige, 2005 & 2010). The
fermented fish formed the principal dietary role in their food habit and hence, they
regarded Southeast Asia as the region of one center of origin of fermented fish
products. Freshwater fish species naturally occurring in local hydrological systems
would have been fermented for preservation (Molinos et al., 2016). In many such
regions with a seasonal flood during monsoon and parchedness during the rest of the
season, the populace exploited the seasonal availability of fish to be preserved for the
lean period through the technique of fermentation (Ruddle &Ishige, 2010)).
2.6 Manufacturing process and diversity of fermented fish
Fermented fish products like fish sauce and fish pastes are regarded as the mother of
all condiments. Fish sauce is produced due to changes in physical and chemical
characteristics by microbial activity in high salt concentrations and low oxygen levels
(Lopetcharat et al., 2001). Traditional and non-traditional fish products are classified,
in different ways, according to the method of processing, substrates used, salt
concentration, mechanism of fermented products, and forms of final products.

10
Traditional and non-traditional fermented foods may be classified into three
categories (Amano, 1962). In the first category, fish fermentation is a natural and
traditional process and the main task is performed by the enzymes present in the fish
flesh and entrails. A little amount of salt is added in this process. In the second
category, 'starter cultures' are used on the flesh of fish and entrails. The ‘starter
culture’ is made of selective microbial enzymes and salt. The third category consists
of non-traditional products manufactured by artificially induced accelerated
fermentation. Saisithi (1987) suggested the classification of traditional fermented fish
products into three groups:
1) first, the carbohydrate source is not used as an additive and the fermented product
is obtained by natural fermentation by the activity of the fish enzyme, bacterial
enzyme already present in fish and salt mixture; 2) Second, the carbohydrate is used
as an additive, and the fish and carbohydrates are fermented by the activity of
bacterial enzymes present in fish and carbohydrate mixture; 3) Third, 'starter culture'
is used that bring about fermentation. Ruddle & Ishige (2005) gave a precise generic
classification and method of preparations of fermented fish in East Asia and classified
the fermented fish into two groups. One consists of fermented fish products from
freshwater fish and the other from marine finfish, shellfish, Crustaceae, etc. Ruddle &
Ishige (2005) classified fermented fish into a high salted product (with 20% salt), low
salt product (with 6-8% salt), and no salt product. Sometimes fermented fishery
products are also categorized according to the form, structure, and composition of the
final product (SubbaRao, 1967; Beddows, 1998). Accordingly, fermented fish are of
three main types:
a. The fermented fish where the original form is retained. Eg: Buro (Phillipines),
makassar (Indonesia), pedah-kemburg (Thailand), makassar (Indonesia),
ngari (India), Colombo cured mackerel (India). Salt is added to the fish and
such type of fermented fish product is also called the salted fish (Beddows,
1998). As the original form of this type of product is retained, the process of
fermentation may or may not take place. Inpedah-siam or pedah-kemburg,
whole fish (Rastrelliger sp., Scomber neglectus and Scomber kangurtal)
which have 15-16% fat with the entrails removed is salted in ratio 3:1(3 fish:
1salt) and stored for 24 hours (Beddows, 1998). The salt desiccates the water
and fish is packed carefully in crates in which the ripening takes place in
anaerobic conditions. ‘Colombo cure’ method is a prevalent practice in

11
 Southern India and Sri-Lanka. In this method usually mackerel fish and salt in
the ratio of 3: 1 are mixed in a concrete tank and dried tamarind fruit is added.
Improvements in the quality are obtained if 5% acetic acid is added in place
of tamarind (Balachandran &Vijayan, 1975; Narzary et al., 2021).

b. Fish sauce: Budu (Malaysia), patis (Philippines), nuoc-mam (Vietnam),

nampla (Thailand) are some of the examples of fish sauce. It is the most well
know product amongst all the fermented fish products in Southeast Asian,
Europe and North America (Narzary et al., 2021). Fish sauce provides a good
amount of daily nitrogen intake, vitamins like vitamin B12 and minerals
(Saisithi, 1994). The fish tissue during fermentation process in fish sauce is
subjected to extensive liquefaction and the resultant fluid part is collected as
the sauce (Kumar & Nayak, 2015; Narzary et al., 2021).
c. Fish paste: Ngapi (Burma), bagoong (Philippines), prahoc (Kampuchea),
trassi (Indonesia) and belachan (Malaysia) are some example of fish paste.
The duration of fermentation in fish paste is generally shorter and the final
product is in paste form (Narzary et al., 2021).

2.7 Fermented fish of Northern Europe

Fermented fish products in Nordic countries like Iceland, Norway, and Sweden are
significant part of their traditional cuisine. Hákarl, rakfisk, surströmming, and Barrel-
salted ripened herring and sprats are the common fermented fish product of Nordic
European countries (Skåra et al., 2015). Hákarl is a delicacy of Iceland prepared by
fermenting Greenland Shark. The meat of Greenland Shark is cut into small pieces
and fermented underground in pits near the sea then hung to dry for several months.
This method of curing reduces the toxicity in Greenland Shark which is otherwise
poisonous when consumed fresh ([Link]
fermented-shark-delicacy/ accesse on 5/3/2020). Rakfisk is produced from arctic charr
and lake trout. It is a Norwegian delicacy and is prepared by salting and fermenting
fish in tight lid containers at low temperature for 3-12 months (Skåra et al., 2015).
Surströmming is a part of Swedish traditional dish. For its production Baltic herring
(Clupea harengus var. membras) found in North Sea of Atlantic ocean is caught
during May to July just before the spawning. The caught are then dipped in saturated
salt solution for 4 hours. After that the fish are beheaded, gutted and put in barrels in
weak salt solution of 17 % (Alm, 2012). For Barrel-salted ripened herring and sprats,

12
Fjord herring, Baltic herring, sprats (Clupea sprattus), anchovies (Engraulis sp.),
sardines (Sardina pilchardus) and different stocks of herring are used. In traditional
method, herrings are beheaded and put in barrels with salt, sugar and some spices
(Timberg et al., 2014; Skåra, 2015).

2.8 Fermented fish products of Africa

Drying, salting and fish fermentation are three artisanal methods used for fish
preservation in African countries. The fermentation process employed in fermented
fish products of Africa is of short duration where the final product retains its original
form and is not transformed into fish paste or sauce (Essuman, 1992). In Ghana
fermented fish is called ‘mamone’ which means stinking fish in Akan language of
Ghana (Nerquaye-Tettch et al., 1978). Guedj, koobi, ewule are the fishery products
that are hard dried or semi-dried and used as fish food that are found in many parts of
Africa. Soft texture fishery products are found in Côte d’Ivoire, Gambia, Ghana and
Sudan which are used as condiment. Feseekh of Sudan is used both as condiment and
fish food (El Sheikha et al., 2014). Some of the popular African fermented fishery
products are ndagala (Barundi), salanga (Chad), guedj, (Gambia, Senegal), djegue
(Mali), fessiekh, terkeen, mideshi, kejeick (Sudan) (Essuman, 1992).

2.9 Fermented fish products of Asia

Because of its unique savor and taste, the popularity of fermented fish in Asian
countries is far reaching (Farnworth, 2008). All three types of fermented fishery
products are found in Asian countries. The fermented fish products found in Thailand
are budu, kapi and pra-la (Phithakpol, 1987). Budu is a popular fermented fish
product with colloidal consistency. Sometimes carbohydrate source is also added in
fermented fish product and Pra-la is such fermented fish paste where rice bran is
added (Rattagool, 1985). The technology used for preparation may be similar, but the
additives are different in different products. The additives rice and fruits are used as
raw materials in these fermented fish products. This allows the growth of fermentative
Lactic Acid Bacteria thereby reducing its pH. Sometimes the similar product may
have divergent names in different localities of Thailand (Narzary et al., 2021). In
Malaysia fermented fish like pakesan is well-liked which is prepared from fresh water
fish mixed with tamarind and salt. Belacan (shrimp paste), cinkaluk, budu and
kikapikan are the diversity of fermented fish products found in Malayasia. In
Phillipines fermented fish products fermented by LAB are consumed (Rattagool,

13
1985). Bagoong (fish paste), balao-balao (fermented shrimps), burongishida
(fermented rice and fish), bagoong alamang (shrimp paste), and paite (fish sauce) are
some of the regular fermented fish product of the Philippine (National Research
Council, 1992). Olympia (1992) reported that many fishes including herring,
anchovie, shrimp etc. and shell fishes that are used for the preparation of bagoong.
Bagoong has a characteristic salty taste, with a faint cheesy odor, and prepared from
fish or shrimp. The fish sauce in Phillipines is called paite and obtained from
thoroughly fermented bagoong. In Indonesia, the popular fermented fish products
are trasi (shrimp paste), pedah (fatty, partly dried, salty fish), jambal roti (fermented
dry salted marine fish), bekasang and bekasam (fermented freshwater fish) budu,
cincaluk, naniura, petis, picungan, pudu, rusip, and tukai (Putro, 1993; Huda, 2012).
Fermented fish products terasi, peda, and kecapikan, contribute to less than 2% of the
total processed fermented fish products (Huda, 2012). In many fermented fish
products of Southeast Asia, koji (Aspergillus sp.) is added to speeden the fermentation
(Steinkraus, 1993). Burma has traditional fermented fish ngapi made from salted fish
or shrimp and is considered the national food of Burma (Steinkraus, 2004). Nuoc-
mam is a fish sauce consumed by a large group of people in Vietnam. It is prepared
from small fishes and shrimps. The fishes are layered with salt and kept for
fermentation. After the aging process, the supernatant liquor decanted off to give first-
quality nuoc-mam (Nghia, 2017). The fish tissue during the fermentation process in
fish sauce is subjected to extensive liquefaction, and the resultant fluid part is
collected as the sauce (Kumar & Nayak, 2015). Yu-lu is a Chinese traditional
fermented fish sauce widely consumed as a condiment for cooking. Anchovies (or the
other small marine fish) are allowed to ferment by adding salt at the level of 30–40%,
salt/fish (w/w) in this product. The period of fermentation is 12–18 months (Jiang et
al., 2007).
In Japan, the fermented fish is called shikora (preserves the whole or partial of
the fish shape), and shikora paste (yielded by pounding or grating). When the
additives like rice, vegetables or salt is used along with fish, then it is known
as narezushi. Shikora and shikora pastes are mostly used as condiments and side
dishes (Ishige, 1993). Korea also possesses a rich dietary tradition of fermented food
and fish. The fermented fish products of Korea are jeotkal, eo-
ganjang, and sikhae (Lee, 1993).

14
2.10 Fermented fish products of India

In the coastal part of India, methods like drying, salting, pit curing and ‘colombo
curing’ are used. Colombo curing is similar to salting and pickling practiced in Sri
Lanka. The practice of pit-curing is common in certain parts of Tamil Nadu and the
neighboring region of Travancore. In this curing method the salted fishes are buried in
pits lined with mat for a period ranging from a few days to a fortnight or more. The
final product is partially dry and possesses a distinct flavour and taste much liked in
the eastern parts of Tamil Nadu (Narzary et al., 2021; Rao et al., 1958). In some
process of curing, Malabar tamarind (Garginia cambogea) is put inside the gut of
gutted and cleaned fish and stacked in barrels containing in brine solution. The fish
curing industry is much neglected in India (Pillai, 1956). However, fermented fish
products are quite popular in North East India and West Bengal. It is interesting to
note that many products do not use salt (Majumdar et al., 2016). In place of salt,
sometimes local alkali called kharwi/khar is used. The type of fermented food
products found in this region is unique and one of a kind (Narzary et al., 2021). The
fermented fish pastes prepared in this region are pounded with various herbaceous
plants like taro and allowed to age inside bamboo stems. Two types of fermented fish
products are reported in North East India: one that retains solid forms after
fermentation and the other in a paste form. They are used as condiments or with other
vegetables for their texture and flavor. The local people associate these products with
some health benefits. Some of the fermented fish products of North East India found
are: a)Shidal is a popular fermented fish product found in different states of North
East India. It is prepared from Puntius sp. (generally Puntius sophore) and Stipinna
phasa and is known by different names across the five states of North East India
(Majumdar et al., 2015) of North East India. b) Ngari is a traditional dry non-salted
fermented fish product of Manipur produced exclusively from sun dried Puntius
sophore Ham. (Jeyaram et al., 2009). Tungtap is traditional fermented fish in
Meghalaya which are prepared from Puntius sp. and Danio sp. (Rapsang and Joshi,
2012). Hentak, numsing, sukakomacha, sukati, napham, nichow, nakhamare some
other fermented fish prepared by different ethnic communities found in North East
India (Thapa et al., 2006; Narzary et al., 2016). Lona ilishis salted and prepared
exclusively from Indian shad (Tenualosa ilisha) popular in both Bangladesh and parts
of India bordering Bangladesh (Majumdar, 2016).

15
2.11 Health Benefits

The advances in scientific studies revealed the significant role of micro-organisms in

fermented food and fish industries, particularly of LAB, with novelties and added
values. LAB isolated from 11 fermented fish samples obtained from the Northeastern
(Sanriku) Satoumi region of Japan showed antioxidant activities and the isolates were
identified as Lactobacillus plantarum, Lactococcus lactis and Pediococcus
pentosaceus (Kuda et al., 2014). LAB, Bifidobacteria and many species of bacteria
are conferred as probiotic. The EPS produced by probiotics enhances nutritional
value, texture, shelf life of the food product and promote gut health ( Korakli et al.,
2002).The studies show that fermented fish also consists of several essential amino
acids like arginine, isoleucine, leucine, lysine, methionine, histidine, phenylalanine,
threonine, tryptophan, and valine in significant amount (Ijong & Ohta,1995; Dincer,
2010).Taurine was found in fermented sea fish of which has the ability to fight
oxidative stress (Martínez-Álvarez et al., 2017). Chai et al., 2020 gave a
comprehensive review on the bioactive peptides from fermented fish. Several
bioactive peptides from fermented fish have been reported to have antioxidative, ACE
inhibitor, antihypertensive, anti-proliferative effect against human lymphoma cell
(Xiang et al., 2019). Fermentation of fish produces desirable organoleptic properties
and increases the bioavailability of minerals (ùanlier et al., 2019). Jemil et al. (2016)
reported 33 biopeptide compounds by fermenting Zebra blenny (Salaria blesilisca)
muscle proteins with stater organism Bacillus mojavensis A21 strain. Fermented fish
contained significantly less fatty acid as compared to fresh fish (Dincer et al., 2010).
Another study showed that fermented fish oil contains notably high levels of EPA
(eicosapentaenoic acid) and DHA (docosahexaenoic acid) (Han et al., 2012). Han et
al. (2012) reported that fermented fish oil contains DHA (docosahexaenoic acid)
which can alleviate the symptoms of atopic dermatitis. Some studies suggested that
bioactive metabolites (e.g. lactoferrin and flavonoids) and polysaccharides from raw
materials containing their precursors are produced in fermented fish (Ryan et al.,
2011).

16
2.12 Health concern

One of the important processes found in fermented fish is Lipid oxidation. But
sometimes because of lipid oxidation the spoilage of fermented fish also take place
and compounds like hexanal, 4-hydroxy-nonanal, valeraldehyde and malondialdehyde
are produced (Feng et al., 2021). These compounds were reported as carcinogenic and
mutagens (Feng et al., 2021). Biogenic amines are nitrogenous compounds consisting
of amine groups that are found in many fermented fish and is considered as the
compounds which are of safety concern (Santos, 1996). The Biogenic amines are
formed by decarboxylation of amino acids and by amination of aldehydes and ketones
and can cause serious health implications when consumed in higher concentration
(Stratton, 1991). BA like histamine and tyramine increases the blood pressure in
sensitive people as they are vasoactive compounds. It may show the symptoms of
allergy, headache, respiratory disorder, nausea and tachycardia (Durak-Dados et al.,
2020). Moon et al., (2010) High concentration of histamine &tyramine was reported
in 47 samples of fermented fish prepared from anchovy, squid, clam and shrimp.
Escherichia coli, Salmonella, Closteridium botulinum, Salmonella sp., Listeria sp.,
and Staphylococcus aureus were the main food born pathogen found in fermented fish
that may show various symptoms of food poisoning (Adams, 2013).

2.13 Biochemical composition of fermented fish products

Fermented fish are alkaline food products that release ammonia during the natural
hydrolysis of proteins into peptides and amino acids, because of which the final pH in
the product increases (Sarkar &Nout, 2014). Alkaline pH and ammonia in these
products control the growth of a few dominant bacteria, which allows the anaerobic
breakdown of proteins that release amine compounds (Narzary et al., 2021). The
fermented fish product is well preserved and stable because of its high pH and free
ammonia. The rapid growth of essential microorganisms in such food does not allow
the growth of bacteria that may spoil the product (Wang & Fung, 1996; Sarkar
&Nout, 2014). Most alkaline fermentations are achieved spontaneously by mixed
bacteria cultures, principally dominated by Bacillus subtilis (Wang & Fung, 1996).
The Physico-chemical characterization of different types of fermented fish depends on
various factors like the nature of processing fish used, percentage of salt
concentration, number of days of fermentation, microorganisms present in the
product, etc. (Narzary et al., 2021).

17
 The biochemical characteristics of pedah, a fermented fish of Indonesia
prepared from Mackerel (Rastrelliger brachysoma), consist of moisture content of
46g, a protein content of 28g, fat 4g, Ca 174mg, and P316mg out of 100g sample
(Irianto & Irianto, 1998).
Jeotgal, a fermented fish of Korea, is prepared from fish Engraulis japonicas
(anchovy) or Ammodytes spp. It consists of moisture content 7.6 %, crude protein
7.4%, crude fat 1.1% carbohydrate 0.01 % and pH 6 %.
Budu is a fermented fish prepared from fish Clupeoides spp., Sardinella spp.,
and Anchoviella commersonii. The moisture content of budu is 45-60.2 %, crude
protein of 12.1-3.2 %, crude fat 1.3-1.8 %, fiber 0.2%-0.4 %, the acidity of 0.4 %-0.9
% and pH 5.4-6.5 % (Saishithi et al., 1994; Phithakpol, 1993).
In ngapi (fermented fish of Myanmar), the moisture content found was 7.6 %,
protein 41.9 %, Fat 8.4 %, NaCl 37.95 % and ash content 3.45 % (Tyn, 1993).
In ngari (India) the raw materials are Puntius sophore and Puntius ticto and
biochemical composition consisted of 36.03 % moisture content, lipid content 13.34
%, ash content 5.49, pH 6.74 %, free fatty acid 97.60 % & water soluble Nitrogen
3.62% (Sarojinalili & Suchitra, 2009).
Shottsuru, a fermented fish sauce in Japan is prepared from anchovy, and
opossum shrimp, wherein salt is used as raw material. The biochemical composition
consisted of Nitrogen 30 to 1598mg/100ml, pH 4.54 to 5.56 %.
Interkin (Sudan), the moisture content consisted of 40 %, protein of 23.8 %,
and pH of 6.1. The raw materials of terkin consisted of fish
Hydrocynus spp., Alestes spp., tilapia, and Nile perch Sardines (Sardinella sp.) or
anchovies (Stelophorus sp.), salt, mustard (Brassica juncea) and water (Abu-Hassan
& Adam Sulieman, 2011; Mohammed, 2010).
Momone (Ghana) has raw materials Barraccuda sp., catfish, croaker, grouper,
herrings, mackerel, octopus, snapper, squid, threadfin, seabream, and ribbon fish
(Anihouvi, 2012), and biochemical composition of moisture content50%, protein16.8-
21.9 %, free fatty acid20.8 %, pH: above 6.0 and NaCl:5.7 % ( Nerquaye Tettch et al.,
1978; Yankah, 1988; Sanni et al., 2002). Mizutani et al. (1991) and Saisithi (1994)
reported the presence of amino acids both essential and non-essential amino acids in
fermented fish products. Glutamic acid and aspartic acid were the dominant amino
acid in rusip, a fermented fish from Indonesia. Other amino acids found in rusip were

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serine, histidine, glycine, threonine, arginine, alanine, tyrosine, methionine, and valine
(Koesoemawardani, 2018). In hatahata-zushi, a Japanese fermented fish, the fatty
acid profile consists of Eladic acid (C18:1n-9), Palmitic acid (C16:0),
Decosahexanoic acid (C22:6n-3), Ecosapentanoic acid (C20:5n-3), and PalC16:1n-7
(Chang, 1994). The various literatures suggest that the production of flavor and
texture of fermentation fish depends on the amino acids and fatty acids that have been
formed after microbial decomposition.

2.14 Microbiota of fermented fish products

Several micro-organisms were reported from different fermented fish products.

Micro-organisms are the integral part of all fermented food products and most of them
are approved by FDA as GRAS (Burdock & Carabin, 2004).
In budu, the strains of bacteria isolated were
Lactobacillus plantarum LP1,
LP2, Staphylococcus arlettae SA1, Saccharomyces cerevisiae SC3 and Candida
glabrata CG2 (Yuen et al., 2016), Bacillus licheniformis, Tetragenococcus
halophilus, Staphylococcus piscifermentans (Choorit & Prasertsan, 1992;
Tanasupawat & Komagata, 1995).
In pedah, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli,
Bacillus cereus, Lactobacillus plantarum and Pediococcus pentosaceus (Indriati et al.,
2006) were reported.
In nauc-man, a fermented fish of Vietnam, Staphylococcus sp.,
Bacillus sp., Pseudomonas sp., Pediococcus sp., Lactobacillus sp., Streptococcus sp,
Halobacterium cutitrubrum, Micrococcus sp., and Halobacterium salinarium were
isolated (Thongthai et al., 1992; Lopetcharat et al., 2001).
In ngapi, Tetragenococcus halophilus, Staphylococcus epidermidis,
Tetragenococcus muriaticus, Halanaerobium fermentans, Tetragenococcus
muriaticus, Clostridium sensuaponi, Clostridium botulinum, Halanaerobium
fermentans ( Kobayashi et al., 2016) were reported.
In shidol, Staphylococcus aureus, Micrococcus sp., Bacillus sp. And
Escherichia coli (Muzzadadi & Basu, 2015) were reported.
In mamone, Aspergillus, Bacillus, Debaryomyces, Hansenula, Lactobacillus,
Pediococcus, Pseudomonas, Staphylococcus and Klebsiella; Bacillus is predominant
with occurrence of 37.7% (Sanni et al., 2002).

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 Bacillus sp., Staphylococcus sp., Lactobacillus sp., Micrococcus sp. plays a
specific role and is present in most of the fermented fish products. In fermented fish
products, factors like use of raw materials and the manufacturing process influence
the type and number of micro-organisms present in them. In some cases, the addition
of certain micro-organisms as 'starter culture' led to the acceleration in fermentation
time, and production of better product (Sumague & Mabesa, 1994). Sumague &
Mabesa, (1994) reported the addition of proteolytic enzymes of Aspergillus oryzae
and Aspergillus niger hastened the production of bagoong and patis. Yongsawatdigul
et al., (2007) reported Staphylococcus sp. SK1-1-5 and Virgibacillus sp. SK 33
strains, when used as 'starter culture' increased the desired volatile compounds in the
fish sauce by increased production of proteinases. Thongsanit et al., (2002) elucidated
that Tetragenococcus halophilus improved the flavor of fish sauce. It produced
volatile compounds like benzaldehyde, 2 &3- methylbutanal, and 2-methylpropanol
that gave the desired flavor to the fish sauce. Fungi like Aspergillus luchuensis
was attributed in generating flavor due to their proteolytic and lipolytic activities in
low salted fermented fish in the study by Kim et al. (2016). Thus, the endogenous
enzymes present in a diverse group of microbes found in fermented fish products can
give desirable organoleptic properties and extended shelf life. The balanced
metabolites present in the fermented fish can determine the preferable flavor and
texture (Jeong et al., 2013). Furthermore, the micro-organisms also degrade the anti-
nutritional and produce antimicrobial properties. Histamine is a hazardous amine
reported in several fermented fishery products that has serious health implications. In
a study by Kuda et al. (2012), Tetragenococcus halophilus isolated from nukazu had
suppressive effect on histamine accumulated on salted fermented fish. Weisella strains
from plaa-som produced antibacterial compounds and folate (Deatraksa et al., 2018).

2.15 Methodologies used for characterizing the microbiota in fermented fish

The microbial communities in fermented food consist of multi-species consortia

involved in the process of biotransformation during fermentation (Babaei, 2019).
Humans over the thousand years could optimize the conditions to support the growth
of specific microbial communities. These microbial communities carry-out different
functions that are fundamental to the texture, flavor, aroma, and safety of fermented
fish (Gomes et al., 2009; Liang et al., 2016). Manipulation of different growth
parameters prerequisite for the microbes, such as moisture, temperature, pH, salinity,

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and chemical composition of the substrate, results in a wide variety of fermented fish
products (Owens et al., 1985). The analysis of microbial communities, their
successional dynamics and, interactions are some of the important features to
understand the process involved in fish fermentation that determines the functionality
and nutritional quality of the final product (Ha et al., 2014). Broadly there are two
approaches to analyze the microbial communities in fermented fish: Culture
dependent method and Culture-independent method. Culture dependent method is a
traditional method of microbial diversity in natural habitat that has been used by
microbiologist for more than 150 years. The cultivable microbes in laboratory
environment are cultured using agar or broth media of selective and enriched nature.
The morphological and physiological characters of the isolated microbes are then
attempted to describe and place the bacteria in taxonomic ranks (Fry, 2004). The
quantification of microbial population is done by counting the serially diluted plate
assay in a unit known as colony forming unit (CFU) and in chambers of optical
microscopy. More recently due to advent of technological revolution sophisticated
microscopy like TEM and Flow cyclometry are also used for quantification (Nguyen,
2017; Fung, 2013). The traditional phenotypic characters are often not reliable for
proper phylogenetic study of isolated bacteria or fungi. Molecular techniques like
sequencing of 16S rRNA and ITS inter-spacer region are nowadays used for
molecular phylogenetic characterization of isolated bacteria and fungus respectively
(Millar et al., 2007). The traditional culture based has limitations in providing some
accuracy in total enumeration of microbes and is very resource as well as time
consuming. Moreover, only the cultivable bacteria can be analyzed by culture based
method which is estimated to be only 1% of the total microbial population of
fermented food (Justé et al., 2008). To overcome these limitations culture-
independent based molecular tools and technology is used. Metagenomics is one such
approach in a genomic analysis that studies the community of micro-organism from
environmental samples without the necessity of obtaining pure cultures (Ranganathan,
2019). It is the process by which metagenome is generated and involves sequencing of
all DNA extracted from the sample followed by sequencing, mapping them to a
reference database and annotation of genes (Lindon et al., 2018). Metagenomics has
dramatically transformed the understanding of microbial diversity. Whole-genome
metagenomics also promises to explore the functionality of microbial consortia in the
fermentation process of food. Libraries prepared from DNA extracted from natural

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samples as well as food samples generate billions of nucleotide sequences.
Techniques of High throughput sequencing or NGS have been used in place of
traditional Sanger sequencing. Sanger sequencing with its limited throughput and high
cost has limited applications in personal genome sequencing (Reuter et al., 2015). The
rise of NGS technologies about a decade ago revolutionized the field of microbiology.
This analyses a comprehensive description of microbial DNA content in a given
sample by generating up to 109 sequences per reading per run (Kulski, 2016). In
commercial hongeo product, a traditional Korean fermented fish Shotgun
metagenomics approach was used to reveal the bacterial community and metabolic
pathways. Four dominant bacterial genera Psychrobacter, Clostrodium, Oblitimonas
and Pseudomonas were detected and alkaliphilic LAB belonging to genera Marini
lactibacillus and Jeotgalibaca were also detected indicating that traditional fermented
food can be good source for extracting novel bacteria (Zhao & Eun, 2020).

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