World Journal of Microbiology & Biotechnology 20: 599–607, 2004.

599

2004 Kluwer Academic Publishers. Printed in the Netherlands.

Microbial diversity in ngari, hentak and tungtap, fermented fish products

of North-East India

Namrata Thapa1, Joydeb Pal2 and Jyoti Prakash Tamang3,*

1
Department of Zoology, Sikkim Government College, Gangtok 737 102, Sikkim, India
2
Department of Zoology, North Bengal University, Siliguri 734 430, West Bengal, India
3
Food Microbiology Laboratory, Sikkim Government College, Gangtok 737 102, Sikkim, India
*Author for correspondence: Tel.: +91-3592-231053, Fax: +91-3592-222707, E-mail: jyoti_tamang@hotmail.com

Received 27 August 2003; accepted 23 January 2004

Keywords: Fermented fish, hentak, microbial diversity, ngari, tungtap

Summary

Ngari, hentak and tungtap are traditional fermented fish products of North-East India. Eighteen samples of ngari,
hentak and tungtap were collected and were analysed for microbial load. Lactic acid bacteria, endospore-forming
rods, yeasts and aerobic mesophilic counts ranged from 4.0 to 7.2, 3.3–4.6, <1–3.5 and 4.3–7.3 log c.f.u./g,
respectively. Lactic acid bacteria were identified as Lactococcus lactis subsp. cremoris, Lactococcus plantarum,
Enterococcus faecium, Lactobacillus fructosus, Lactobacillus amylophilus, Lactobacillus coryniformis subsp. torquens
and Lactobacillus plantarum. Endospore-forming rods were identified as Bacillus subtilis and Bacillus pumilus,
aerobic coccal strains were identified as Micrococcus. Yeasts were identified as species of Candida and
Saccharomycopsis. Pathogenic contaminants were detected in all samples, however, none of the sample contained
more than 102 c.f.u./g of Bacillus cereus, 103 c.f.u./g of Staphylococcus aureus and enterobacteriaceae population,
respectively. Enzymatic and antimicrobial activities of the isolates were tested. None of the strains produced
biogenic amines in the method applied. Most strains of LAB had a high degree of hydrophobicity, indicating their
‘probiotic’ characters. This study has demonstrated the microbial diversity within the species of lactic acid bacteria,
Bacillus and yeasts.

Introduction 1988). Tungtap is a fermented fish paste, commonly

consumed by the Khasia tribes of Meghalaya in North-
Traditional processing of fish such as fermentation, East state of India (Thapa 2002). Dry fish (Danio spp.) is
salting, drying and smoking are the principal methods of mixed with salt, kept in an earthen pot and fermented for
fish preservation in South-East Asia (Cooke et al. 1993). 4–7 days. It is consumed as a pickle.
Ethnic people of North-East India catch fishes from the There appears to be no information on the microbio-
rivers and lakes, some of these are traditionally fer- logy of these traditionally processed fish products of
mented (Tamang 2001). Ngari is a fermented fish product North-East India. The aim of the present study was to
of Manipur in North-East India. During its preparation, examine the composition of microorganisms, mainly the
the fish (Puntius sophore) is rubbed with salt, dried in the lactic acid bacteria of ngari, hentak and tungtap.
sun for 3–4 days, pressed tightly in an earthen pot, sealed Enzymatic activities, antimicrobial properties, ability
airtight and then stored at room temperature for 4– to produce biogenic amines and degree of hydropho-
6 months (Thapa 2002). Ngari is eaten as a side-dish with bicity of isolates were also examined.
cooked rice. Hentak is a ball-like thick paste prepared by
fermentation of a mixture of sun-dried fish (Esomus
danricus) powder and petioles of aroid plants (Alocasia Materials and methods
macrorhiza) in Manipur (Thapa 2002). Dry fish is crushed
to powder, an equal amount of petioles of aroid plants is Collection of samples
mixed and a ball-like thick paste is made. The mixture is
kept in an earthen pot and is fermented for 7–9 days. Six samples of each ngari and hentak were purchased
Hentak is consumed as curry as well as a condiment with from different shops in Ima market of Imphal in
boiled rice. Sometimes, it is given to mothers in confine- Manipur, respectively, and six samples of tungtap were
ment and patients in convalescence (Sarojnalini & Singh collected from Shillong of Meghalaya. Samples were
600 N. Thapa et al.
collected aseptically in pre-sterile poly-bags kept in an bacterial strains were observed in a phase contrast
ice-box, and transported to laboratory for analyses. microscope (Olympus CH3-BH-PC, Japan) following
the method of Harrigan (1998). Gram staining of
Microbial analysis isolates was performed following the method of
Bartholomew (1962). Catalase test was studied using
Ten gram of sample was suspended in 90 ml of 0.85% 0.5 ml of 10% hydrogen peroxide solution and observed
(w/v) sterile physiological saline and homogenized in a for the production of gas bubbles. Isolates were iden-
stomacher lab-blender 400 (Seward, UK) for 1 min. tified by phenotypic properties using CO2 production
Decimal dilution series were prepared in sterile diluent from glucose, growth at different temperatures, pH and
and diluted suspension of sample was mixed with the ability to grow in varying concentrations of NaCl in
molten media and poured into plates. Lactic acid MRS broth as described by Schillinger & Lücke (1987)
bacteria (LAB) were selectively isolated on MRS agar and Dykes et al. (1994). The configuration of lactic acid
(HiMedia M641, India) plates supplemented with 1% produced was determined enzymatically using D -lactate
CaCO3 and incubated under anaerobic conditions in an and L -lactate dehydrogenase kits (Roche Diagnostic,
Anaerobic Gas-Pack system (HiMedia LE002, India) at France). The presence of meso-diaminopimelic acid
30 C for 3 days. The predominant LAB were obtained (m-Dpm) in the cell walls of lactic acid bacteria was
from MRS plates with the highest sample dilutions (10)5 determined using thin chromatography on cellulose
or 10)6). For aerobic endospore bacterial counts, a plate (Schillinger & Lücke 1987). Ability of the isolates
diluted suspension of sample was heated for 2 min in to ferment carbohydrates was studied using API 50
continuously boiling water (Tamang & Nikkuni 1996). CHL (bioMérieux, France) system. Bacterial strains
Bacterial endospores were enumerated in pour-plates of were identified following the taxonomic keys laid down
nutrient agar (HiMedia MM012, India) and incubated in Bergey’s Manual of Systematic Bacteriology, volume 2
aerobically at 37 C for 1 day. Aerobic mesophilic (Sneath et al. 1986) and Wood & Holzapfel (1995).
counts (AMC) were determined using plate count agar Endospore-forming bacteria were identified according
(HiMedia M091A, India) incubated at 30 C for 2 days. to the keys based on Claus & Berkeley (1986) and
Moulds and yeasts were isolated on potato dextrose Slepecky & Hemphill (1992).
agar (HiMedia M096, India) and yeast extract-malt
extract agar (HiMedia M424, India), respectively sup- Proteolytic activity
plemented with 10 IU/ml benzylpenicillin and 12 lg/ml
streptomycin sulphate and incubated aerobically at Surface-dried plates of milk agar (Gordon et al. 1973)
28 C for 3 days. Colonies were selected randomly or were streaked with 24-h-old cultures, incubated at 30 C
all sampled if the plate contained less than 10 colonies, for 4 days (lactic acid bacteria) and 37 C for 2 days
according to Leisner et al. (1997). Purity of the isolates (endospore-forming bacteria), and examined for any
was checked by streaking again on fresh agar plates of clearing of casein around and underneath the growth for
the isolation media, followed by microscopic examina- assessment of proteolytic activity.
tions. Identified strains of microorganisms were pre-
served in 15% glycerol at )20 C and deposited at Amylolytic activity
Culture Collection Centre of Food Microbiology Lab-
oratory, Sikkim Government College, India. Surface-dried plates of starch agar (Gordon et al. 1973)
were streaked with 24-h-old cultures, incubated at 30 C
Pathogenic contaminants for 4 days (lactic acid bacteria) and 37 C for 2 days
(endospore-forming bacteria). After incubation the
Ten gram of sample was blended with in 90 ml of plates were flooded with iodine solution for 15–30 min
peptone–physiological saline (0.1% neutral peptone, and examined the clear zone underneath (after the
0.85% NaCl) in a stomacher lab-blender 400 (Seward, growth was scrapped off) for amylolytic activity.
UK) for 1 min. Serial dilution series were prepared in
the same diluent in duplicates. Samples were tested for Lipolytic activity
enumeration of pathogenic contaminants such as Bacil-
lus cereus using selective Bacillus cereus agar base Surface-dried plates of tributyrin agar (Leuschner et al.
(HiMedia M833, India), Staphylococcus aureus using 1997) were streaked with 24-h-old culture and incubated
Baird Parker agar base (HiMedia M043, India) and at 30 C for 4 days (lactic acid bacteria) and 37 C for
enterobacteriaceae using Violet Red Bile Glucose agar 2 days (endospore-forming bacteria). Lipolytic activity
(HiMedia M581, India) (Han et al. 2001). was detected by a clear zone surrounding the culture in
the turbid tributyrin agar.
Characterization and identification
Protease activity assay
Characterization and identification of yeasts were car-
ried out following the method described by Kurtzman & Protease activity was measured by a modification of the
Fell (1998). Cell morphology and motility test of method of Maeda et al. (1993). Cultures were grown in
Microbial diversity in fermented fish products 601
phytone broth (Nagai et al. 1994) on a rotary shaking Streptococcus mutans DSM 6178. The bacteriocin activ-
incubator at 30 C at 180 rev/min for 72 h. Cultures ity of the isolates was determined using cell-free neu-
were immediately centrifuged at 17,000 rev/min for tralized supernatants (Uhlman et al. 1992).
10 min. The enzyme solution was diluted to an appro-
priate concentration. The enzyme solution and the
Biogenic amine
substrate solution containing 1% Azocasein (Sigma
Chemical Co., USA) was dissolved in 100 mM phos-
The ability of functional LAB to produce biogenic
phate buffer, (pH 6.8) were pre-incubated separately at
amines was determined qualitatively on an improved
37 C for 5 min in a water-bath incubator (RSB-12,
screening medium as described by Bover-Cid & Hol-
Remi, India). The enzyme reaction was started by
zapfel (1999) using a ‘cocktail’ of four precursor amino
adding 2 ml of 1% Azocasein to 1 ml of enzyme solution
acids (histidine, lysine, ornithine and tyrosine).
and incubated at 37 C for 20 min. The reaction was
quenched by the addition of 2.5 ml of 10% (w/v)
trichloroacetic acid. After centrifugation at 15,000 rev/ Hydrophobicity
min for 10 min, 2 ml of supernatant was neutralized
with equal amount of 1 N NaOH and the absorbance The degree of hydrophobicity of the strains was deter-
was measured at 450 nm in UV–VIS spectrophotometer mined by employing the methods described by Rosen-
(Specord 200, Analytik Jena, Germany). One unit of berg (1984) and Ding & Lämmler (1992). These methods
protease activity was defined as the quantity required to were based on adhesion of cells to hexadecane droplets.
increase the absorbance by 0.1 under the above condi- Cultures were grown in 5 ml of MRS broth, centrifuged
tions. at 7500 rev/min for 5 min and the cell pellet was washed
with 9 ml of Ringer solution (Merck), resuspended in a
a-Amylase activity assay cylomixer and repeated twice. The 1 ml of the suspension
was taken and the absorbance at 580 nm was measured
The blue value method of Fuwa (1954) as modified by in UV–VIS spectrophotometer (Specord 200, Analytik
Kawaguchi et al. (1992) was followed for determination Jena, Germany). Then 1.5 ml of the suspension was
of a-amylase activity. Cultures were grown on broth mixed with 1.5 ml of n-hexadecane (HiMedia RM 2238,
medium (1.0% soluble starch, 1.0% beef extract, 1.0% India) in duplicate and mixed thoroughly in a cylomixer
peptone, and 0.3% NaCl, pH 7.0) on a rotary shaking for 2 min. The two phases were allowed to separate for
incubator at 30 C at 180 rev/min for 48 h. The cultures 30 min. The 1 ml of the lower phase was taken and the
were immediately centrifuged at 17,000 rev/min for absorbance at 580 nm was measured. The percentage
10 min. The enzyme solution was diluted to an appro- hydrophobicity of strain adhering to hexadecane was
priate concentration. The enzyme solution and 1.5% calculated using the equation:
soluble starch dissolved in 100 mM Tris–HCl buffer (pH
7.0) were pre-incubated separately at 37 C for 5 min in Hydrophobicity (%)
water-bath shaker (RSB-12, Remi, India). Then, the OD580 ðinitialÞ
OD580 ðwith hexadecaneÞ  100
¼
reaction mixture was started by adding 1 ml of 1.5% OD580 ðinitialÞ
soluble starch (HiMedia RM089, India) to 0.5 ml
enzyme solution and incubated at 37 C for 10 min. Replications
The reaction was stopped by the addition of 2.5 ml of
stop solution (0.5 M acetic acid–0.5 M HCl, 5:1). Then All samples were analysed using duplicate subsamples.
100 ml of the reaction mixture was added to potassium Microbiological data were calculated from two to four
iodide solution, left at room temperature for 20 min and counting plates for each subsample and microorganism,
the absorbance at 660 nm of the resulting solution was and reported as mean values.
measured in UV–VIS spectrophotometer (Specord 200,
Analytik Jena, Germany). One unit of a-amylase activity
was defined as the amount of a-amylase which produced Results
10% reduction in the intensity of blue colour under the
above conditions. Microorganisms

Antagonism and bacteriocin activity Six samples of each ngari, hentak and tungtap were
analysed for microbial load, respectively (Table 1). The
Isolates were screened for antagonistic activity by the load of lactic acid bacteria (LAB) was higher than that of
deferred inhibition test described by Schillinger & Lücke other microorganisms in all three fermented fish prod-
(1989), using the bacteriocin screening medium ucts. Mould was not recovered in any sample analysed.
(Tichaczek et al. 1992). The indicator strains used for Out of 121 LAB strains isolated from ngari, hentak and
antagonist as well as bacteriocin screening included: tungtap, 66 strains were cocci and 55 strains were
Listeria monocytogenes DSM 20600, Bacillus cereus non-sporeforming rods. All strains of LAB were
CCM 2010, Enterococcus faecium DSM 20477 and Gram-positive, non-sporeformers, non-motile, catalase-
602 N. Thapa et al.
Table 1. Microbial counts of fermented fish products. a-amylase and lipolytic activities, respectively (Table 5).
Product Log c.f.u./g sample
LAB showed low protease activity whereas Bacillus
subtilis showed remarkable high activity. Antagonistic
LAB Bacterial Yeast AMC properties of LAB and Bacillus isolates were tested
endospores against the indicator strains (Listeria monocytogenes
Ngaria 6.8 4.2 3.1 7.0 DSM 20600), Bacillus cereus CCM 2010, Enterococcus
(5.8–7.2) (3.3–4.6) (2.8–3.3) (6.3–7.2) faecium DSM 20477 and Streptococcus mutans DSM
Hentaka 4.6 3.8 <1 4.7 6178). Lactobacillus coryniformis subsp. torquens T2:L1
(4.0–5.8) (3.3–4.2) (4.3–5.9) (isolated from tungtap) showed inhibition zone against
Tungtapa 5.9 3.2 3.0 6.1
(5.2–6.2) (2.5–3.7) (2.6–3.5) (5.3–6.3)
Enterococcus faecium DSM 20477 and Bacillus subtilis
T1:S1 (isolated from tungtap) against Streptococcus
c.f.u. – Colony forming unit; LAB – lactic acid bacteria; AMC – mutans DSM 6178. None of the strains were found to
aerobic mesophilic count. produce any bacteriocin with the methods applied (data
a
Data represent the means of six samples. Ranges are given in not shown).
parentheses.

Biogenic amines
negative and facultative anaerobes; they did not hydro-
lyse casein, gelatin and starch. Gas production from
Isolates of LAB and Bacillus spp. were screened for their
glucose was used as a first step in the differentiation of
ability to produce biogenic amines with the surface plate
lactic rods (Kandler 1983). Heterofermentative LAB was
method as described by Bover-Cid & Holzapfel (1999).
identified as Lactobacillus fructosus and homofermenta-
Interestingly, none of the strains produced tyramine,
tive LABs were identified as Lactobacillus amylophilus,
cadaverine, histidine and putrescine in the method
Lactobacillus coryniformis subsp. torquens and Lactoba-
applied.
cillus plantarum (Table 2) on the basis of sugar fermen-
tation using the API system, lactic acid isomer and meso-
diaminopimelic acid determination, and also based on Hydrophobicity
the taxonomical keys of Sneath et al. (1986) and Wood &
Holzapfel (1995). Coccal LABs were identified as Lac- Table 6 shows the percentage hydrophobicity of the
tococcus plantarum, Lactococcus lactis subsp. cremoris LAB isolates. Two strains of LAB showed high degrees
and Enterococcus faecium (Table 2). Forty-six strains of of hydrophobicity (>75%), among which Lactobacillus
endospore-forming rods were isolated from fermented fructosus HL1 (isolated from hentak) showed the highest
fish samples. Based on key of Slepecky & Hemphill percentage of hydrophobicity of 84.3%. All tested
(1992) representing all species of Bacillus described by strains had more than 30% hydrophobicity.
Claus & Berkeley (1986), representative strains of
endospore-forming rods were identified as Bacillus sub-
tilis and Bacillus pumilus. Sixteen strains of aerobic cocci Discussion
were isolated from fermented fish products. All strains
were Gram-positive, cocci in tetrads and clusters, non- Ngari and hentak are unique fermented fish cuisine of
spore-formers, non-motile and catalase-positive. These Manipur. Tungtap is a traditional fermented fish pro-
strains were identified as Micrococcus. However, the duct consumed by the Khasia tribes in Meghalaya in
species could not be identified due to limited tests. North-East India. Lactic acid bacteria were pre-domi-
Sixteen strains of yeasts were isolated from ngari and nant in ngari, hentak and tungtap. LAB species were
tungtap. On the basis of the taxonomical keys of also reported from other Asian fermented fish products
Kurtzman & Fell (1998), yeasts strains were identified such as species of Lactobacillus from nam-plaa and kapi,
as Candida and Saccharomycopsis (Table 3). Yeasts were fermented fish products of Thailand (Tanasupawat et al.
not recovered from hentak samples. 1992). Though the load of Bacillus spp. was around
104 c.f.u./g, their presence shows the dominance in fish
Pathogenic contaminants products next to LAB. Bacillus species were found in the
fish products due to their ability as endospore formers to
Pathogenic contaminants Bacillus cereus, Staphylococcus survive under the prevailing conditions (Crisan & Sands
aureus and enterobacteriaceae were detected in ngari, 1975). Species of Micrococcus were also recovered in a
hentak and tungtap (Table 4). Count of Bacillus cereus few samples of ngari, hentak and tungtap. Species of
was <102 c.f.u./g whereas the number of enterobacteri- Micrococcus have also been reported from some fer-
aceae and Staphylococcus aureus were <103 c.f.u./g. mented fish products of Thailand (Phithakpol 1993) and
Japan (Wu et al. 2000). Yeasts such as Candida and
Enzymatic activity and antimicrobial activity Saccharomycopsis were also present in ngari, hentak
and tungtap. Species of Candida and Saccharomyces
Representative strains of LAB and Bacillus, isolated were also reported from nam-plaa and kapi (Watanap-
from ngari, hentak and tungtap were tested for protease, uti et al. 1983).
Table 2. Characteristics of lactic acid bacteria strains isolated from fermented fish products of Northeast India.
Microbial diversity in fermented fish products
603
 604

Table 3. Characteristics of yeasts isolated from fermented fish products of Northeast India.
N. Thapa et al.
Microbial diversity in fermented fish products 605
Table 4. Microbial counts of pathogenic contaminants in fermented Table 6. Percentage hydrophobicity of LAB strains isolated from
fish products. fermented fish products.

Product Log c.f.u./g sample Product Strain % Hydrophobicity

Bacillus cereus Staphylococcus Enterobacteriaceae Ngari Lactococcus plantarum Ng1:R2 60.0 (+)
aureus Lactococcus plantarum Ng2:L4 67.2 (+)
Lactococcus plantarum Ng2:L5 53.0 (+)
Ngaria 2.3 3.0 3.3
(1.8–2.6) (2.5–3.5) (3.1–3.5) Hentak Lactobacillus fructosus HL1 84.3 (++)
Hentaka 2.2 2.8 3.0 Lactobacillus amylophilus H1:B1 55.3 (+)
(1.7–2.4) (2.4–3.0) (2.7–3.3) Enterococcus faecium H2:B3 45.6 (+)
Tungtapa 2.3 <1 3.5 Tungtap Lactobacillus coryniformis subsp. 63.7 (+)
(1.7–2.5) (2.9–3.9) torquens T2:L1
Lactococcus lactis subsp. 47.1 (+)
a
Data represent the means of six samples. Ranges are given in cremoris T2:L2
parentheses. Lactobacillus fructosus T2:L5 81.0 (++)

++ – Hexadecane adherence ‡75% (hydrophobic); + – hexade-

cane adherence 26–74% (intermediate).
Population of Bacillus cereus, Staphylococcus aureus
and enterobacteriaceae was not more than 103 c.f.u./g in
samples of ngari, hentak and tungtap, which would be tissues (Backhoff 1976). Lactobacillus coryniformis
the impact of competition and/or antagonistic reaction subsp. torquens T2:L1, isolated from tungtap, showed
of predominant lactic acid bacteria that have prevented antagonistic properties against the indicator strains,
the proliferation (Adams & Nicolaides 1997). However, which can reduce the number of other undesired
the presence of Bacillus cereus, Staphylococcus aureus microorganisms in the fish products as well as help in
and enterobacteriaceae in fermented fish products was the preservation of fish (Einarsson & Lauzon 1995).
due to contamination during processing. A small num- Biogenic amines have been reported in fish products
ber of Bacillus cereus counts in foods is not considered (Ten Brink et al. 1990). None of the isolates tested was
significant (Beumer 2001). Staphylococcus aureus is found to decarboxylase the amino acids used tyrosine,
regarded as a poor competitor and its growth in lysine, histidine and ornithine. However, the lack of
fermented foods is generally associated with a failure histamine, tyramine, cadaverine and putrescine produc-
of the normal microflora (Nychas & Arkoudelos 1990). ers isolated from traditionally fermented fish products in
Though there has been no reported case of toxicity or our study could possible be explained by the lack of free
illness due to consumption of fermented fish products in amino acids within the samples. The concentration of
North-East India, gross contamination of fermented fish amino acids in food is important for biogenic amines
products in the region suggests that the products need to formation (Joosten & Northolt 1989). Before confirming
be investigated clinically. the non-production of biogenic amines in ngari, hentak
All isolates of LAB showed low protease activity, and tungtap, qualitative and quantitative analysis of
whereas isolates of Bacillus strains showed remarkable biogenic amine is necessary.
proteolytic activities. Some isolates of LAB showed high Adherence is one of the most important selection
amylolytic activity. Proteolysis and liquefaction that criteria for probiotic bacteria (Shah 2001). Strains of
occur during fish production has been reported to be LAB isolated from ngari, hentak and tungtap showed
largely the result of autolytic breakdown of the fish high degrees of hydrophobicity indicating that the

Table 5. Enzymatic activity of the selected strains, isolated from fermented fish products.

Product Strain Proteasea (U/ml) a-Amylasea (U/ml) Lipolytic activity

Ngari Lb. plantarum Ng1:R2 0.5 1.1 )

Lactococcus plantarum Ng2:L4 0.5 1.0 )
Lactococcus plantarum Ng2:L5 0.4 4.4 +
Bacillus subtilis Ng1:S1 4.3 1.2 +
Bacillus pumilus Ng1:S2 1.0 0 )
Hentak Lb. fructosus HL1 0.3 1.0 )
Lb. amylophilus H1:B1 0.3 0.9 )
Enterococcus faecium H2:B3 0.8 1.2 +
Bacillus subtilis H1:S1 4.5 2.3 +
Tungtap Lb. coryniformis subsp. torquens T2:L1 1.0 1.1 )
Lactococcus lactis subsp. cremoris T2:L2 0.4 3.1 )
Lb. fructosus T2:L5 0.3 1.1 +
Bacillus subtilis T1:S1 2.7 3.2 +

a
Strains showing positive hydrolysis test (>2.0 mm) were assayed. Data represent the means of three sets. Lb – Lactobacillus.
606 N. Thapa et al.
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