Journal of Scientific & Industrial Research

Vol. 65, September 2006, pp. 747-752

Microbial and analytical characterization of Chhu-_.A traditional fermented milk
product of the Sikkim Himalayas
Sailendra Dewan and Jyoti Prakash Tamang*
Food Microbiology Laboratory, Department of Botany, Sikkim Government College, Gangtok 737 102
Received J7 November 2005; accepted J7 May 2006
Chhu, a traditional fermented milk product prepared from cow milk or yak milk in Sikkirn, India, contains: lactic acid
bacteria (LAB), 8.1-8.8; yeasts, 6.0-6.9; and total viable counts, 8.9-9.2 Log cfu/g, No mould was detected. LAB were
identified as Lactobacillus alimentarius, Lb. farciminis, Lb. salivarius, Lb. bifermentans, UJ. brevis and UlctOCOCCllS lactis
subsp. cremoris, all of which showed a high degree of hydrophobicity, suggesting a possible probiotic character. Yeasts were
identified as Saccharoniycopsis crataegensis and Candida castellii. LAB produced a wide spectrum of enzymes. None of the
strains produced bacteriocin and biogenic amines. Most strains of LAB coagulated skim milk with a moderate drop in pH. A
proximatecompositionofChhu was similar to a typical cheese.
Keywords: Chhu, Fermented milk, Lactic acid bacteria
IPC Code:C12P
Introduction
Dairy products constitute basic diet of ethnic people
of Sikkim Himalayas in India'. Chhu (Sheden) is a
strong-flavoured traditional cheese-like product
prepared from cow milk or yak milk in Sikkim and the
Darjeeling Hills, parts of Arunachal Pradesh, and
Ladakh in India, besides Nepal, Bhutan and China
(Tibet). During its preparation, curd is churned in a
bamboo or wooden vessel, butter-milk is collected and
cooked for 10-15 min to get a soft mass, which is
sieved out and put inside a muslin cloth to drain out the
remaining whey. Soft 111USS is placed in a closed vessel
and is left for natural fermentation for 5-7 days at room
temperature to prepare Chhu (Fig. 1). It is consumed as
curry by cooking in butter along with onions, tomatoes
and chi lies (Fig. 2), and is mixed with beef or yak
meat. Ema dachi, hot-curry of Chhu, is most delicious
food in Bhutan.
This study analyzes microbial profile and
technological properties of predominant functional
microorganisms and a proximate composition of Chhu.
Materials and Methods
Collection of Samples
. A total of 35 samples of Chhu {cow Chhu (CC),
21; yak Chhu (Ye), 14} were collected aseptically
from different places of Sikkim.
*Author for correspondence
Tel: +91-3592-231053
Evmail: jyoti_talnang@hollnail.com
Microbiological Analysis
Samples (10 g) were homogenized with sterile
physiological saline (90 ml) in a stomacher lab-
blender (400, Seward, London,UK) for--I min and
serially diluted in the same diluent. Lactic acid
bacteria (LAB) were isolated on MRS agar (M641,
HiMedia, Mumbai) plates, after incubation under
anaerobic conditions in an anaerobic gas-pack system
(LE002, HiMedia, Mumbai) at 30C for 48-72 h.
Total viable counts were determined using plate count
agar (M091A, HiMedia, Mumbai) incubated
aerobically at 30C for 48-72 h. Aerobic spore-
forming bacteria were isolated on nutrient agar, after
inactivation of vegetable cells by heating at IOOoe for
2 rnin", and were incubated at 37C for 24 h. Moulds
and yeasts were isolated on potato dextrose agar
(M096, HiMedia, Mumbai) andYM agar (M424,
HiMedia, Mumbai), supplemented with 10 IU/m)
benzylpenicillin and 12 ug/rnl streptomycin sulphate,
respectively and incubated aerobically at 28C for
72 h. Samples were tested:' for enumeration of
Bacillus cereus using selective B. cereus agar base
(M833, HiMedia, Mumbai), Staphylococcus aureus
using Baird Parker agar base (M043, Hilvledia,
Mumbai) and enterobacteriaceae using violet red bile
glucose agar (M581, Hilvledia, Mumbai).
Characterization and Identification
LAB isolates were Gram-stained and tested for
catalase production. Cell morphology and motility test
748
J SCI IND RES VOL 65 SEPTEMBER 2006
Fig. l--Cow-ll1ilk Chhu of Sikk im
Fig. 2--Chhu curry
were observed in a phase contrast microscope (CH3-
BH-PC, Olympus, Japan). LAB isolates were
identified" on the basis of phenotypic characters. The
configuration of lactic acid produced was determined
enzymatically using D-lactate and L-Iactate
dehydrogenase kits (Roche Diagnostic, France). The
presence of meso-diaminopimelic acid (OAP) in the
cell walls of LAB was determined using thin-
chromatography'. Sugar fermentation of LAB was
determined using API 50 CHL strips (biolvlerieux.
France). APILAB PLUS database identification
software (biolvlerieux, France) was used to interpret
results. Method" was used to characterize and identify
yeasts.
Enzymatic Profile by API-zym System
Enzymatic profiles of LAB were assayed using
API-zym (biolvlerieux, France) galleries by testing
activity of following 19 enzymes: alkaline
phosphatase, esterase (C4), esterase lipase (C8), lipase
(C14), leucine-, valine- and cystine- arylarnidase,
trypsin, a-chymotrypsin, acid phosphatase, naphthol-
AS-BI-phosphohydrolase, a- and
glucuronidase, a-and
glucosaminidase, a-mannosidase and fucosidase.
Acidification and Coagulation
Acidification and coagulation of milk by LAB
strains was assayed by inoculating 10 % skim milk
(RM1254, HiMedia, Mumbai) at 1 % level and
incubated at 30C and 37C. Observation was made
for commencement of clotting, followed by pH
measurement
7

Antagonism and Bacteriocin Activity

LAB isolates were screened for antagorusuc
activity by the agar spot method
8
against Listeria
innocua DSM 20649, L. monocytogenes DSM 20600,
B. cereus CCM 2010, Staphylococcus aureus SI,
Enterococcus faecium DSM 20477, Streptococcus
mutans DSM 6178, Klebsiella pneumoniae subsp.
pneumoniae BFE 147, Enterobacter cloacae BFE
282, E. agglomerans BFE . 154 and Pseudomonas
aeruginosa BFE 162. Cell-free neutralized
supernatants fluids of LAB isolates were screened for
bacteriocin production by the agar spot test method'),
using the bacteriocin screening medium'",
Biogenic Amine
Ability of LAB isolates to produce biogenic arnincs
was determined qualitatively on an improved
screening medium" using a 'cocktail ' of four
precursor amino acids (histidine, lysine, ornithine,
tyrosine). Change of bromocresol purple indicator to
purple was index of significant amino ac id
decarboxylase activity, corresponding to >350 mg of
a particular amino acid/l ' . .
lIydrophobicity
To determine hydrophobicity of LAB strains
' 2
,
isolates were grown in MRS broth (M369, HiMedia,
Mumbai) at 30C for 24 h and centrifuged at 7,500
DEWAN& TAMANG: MICROBIOLOGY OF'CHHU
749
Table I-Microbial characteristics ofChhu of Sikkirn
Data represent the means of number of samples (n):
Microbiological data were transformed into logarithms of the
numbers of colony fanning unit (cfu)/g; DL, detection limit is
10 Mould was not detected,
Hydrophobicity (%) =
_0_D_5_8_0_(_i n_it_ia_I_)-_0 __D_5_80 __(w_i_tf_l_he_x_a_d_ec_a_n_e)_ x 100
OD580 (initial)
Results and Discussion
Samples of Chhu prepared from cow milk (21) and
from yak milk (14) were analyzed for microbial
counts (Table 1). The population of LAB, yeasts and
the total viable counts was found at the level of
10
8
cfu/g, 10
6
cfu/g and 10
9
cfu/g, respectively.
Bacterial endospores were detected only in samples of
CC. Counts of B. cereus, S. aureus and
enterobacteriaceae were found less than 10
2
cfu/g in
Chhu samples. Mould was not recovered in any
sample.
A total of 120 strains of LAB isolated from Chhu
were grouped randomly on the basis of gas
production, arginine hydrolysis and cell morphology
into representative strains. Based on phenotypic
characterization including lactate configuration, DAP
determination and interpretation of APILAB PLUS
database, and also taxonornical key'", homo-
ferrnentative lactic strain CUG3: RI (CC) was
identified as Lactobacillus alimentarius, strain
CUG1:R2 (CC) as Lb..[arciminis, and strain KCK1:Rl
(YC) as Lb. salivarius; hetero-ferrnentative strain
KCK2:R4 (YC) was identified as Lb. b{lernientans,
and strains CUG2:R2 (CC) and KCYI:R1 (YC) as Lb.
brevis. Coccus strain CUR1: Cl (CC) was identified as
Lactococcus lactis subsp. creinoris. The lactobacilli
were predominant lactic acid flora represented 93 % in
Chhu samples. The identity of LAB seeIns to
correspond with that of LAB typically reported for
milk products16. LAB present in raw cow milk may
contribute to the spontaneous fermentation 17.
Yeast strains (37) were grouped randomly on the
basis of colony, cell morphology, vegetative characters,
and sugar fermentation and assimilation tests.
Following taxonomical keys", yeasts were identified as
Saccharomycopsis crataegensis and Candida castellii.
Presence of high number of yeasts (10
6
cfu/g) indicates
some role during spontaneous fermentation of Chhu,
Yeasts bring about desirable fermentation changes in
fermented milk products",
Enzymatic profiles of LAB strains, assayed using
the API zym (biolvlerieux, France) galleries (Table 2),
showed relatively weak esterase and strong leucine
arylamidase, f3-galactosidase and glucosidase activities,
and no detectable proteinase activity. The use of API-
zym technique has relevance for selection of strains as
potential starter cultures for accelerated maturation and
flavour development of milk products", Absence of
proteinases, and presence of high peptidase (leucine-
arylamidase) and esterase-lipase (C4 and C8) activities
produced by the LAB isolated from Chhu are traits of
desirable quality for their use in production of typical
flavour. High activity of f3-galactosidase exhibited by
LAB species is essential features in Chhu. All LAB
strains except Lb. brevis caused coagulation of milk at
both 30C and 37C with a significant drop in
pH (Table 3). However, coagulation occurred faster
(17-36 h) at 37C than 30C (20-38 h). Coagulation of'
Range of counts (Log cfu/g sample)
Cow-Chhu (n = 21) Yak-Chhu (n = 14)
8.1-8.3 8.5-8.8
<DL-2.7 <DL
<DL-2.8 <DL-2.4
1.7-2.4 <DL-I.5
1.8-2.5 1.2-2.0
6.6-6.9 6.0-6.7
8.9-9.1 8.8-9.2
Microorganisms
Lactic acid bacteria
Bacterial cndospores
Bacillus cercus
Staphylococcus aureus
Enterobacteriaceae
Yeast
Total viable count
rpm for 5 min. The pellet was washed three times
with 9 ml of Ringer solution (Merck, Germany) and
thoroughly mixed in Cl vortex. Suspension (1 ml) was
taken and the absorbance at 580 nm was measured.
Then, 1.5 ml of suspension was mixed with equal
volume of n-hexadecane (RM 2238, HiMedia,
Murnbai) in duplicates and mixed thoroughly. Phases
were allowed to separate for 30 min at room
temperature, after which aqueous phase was carefully
removed and absorbance at 580 nm was measured.
Percentage hydrophobicity was expressed as follows:
Analysis of Proximate Composition
The pH of sample was determined directly using a
digital pH meter (Type 361, Systronics, India).
Titratable acidity, moisture, ash, fat and protein
contents of sample were determined using methods of
AOAC
I 3
Carbohydrate content was estimated by
difference: 100-(% protein + % fat + % ash)".
Calcium, magnesium, manganese, iron and zinc were
estimated in an atomic absorption spectrophotometer
(model 2380, Perkin-Elrner, USA).
750
Enzyme
J SCIIND RES VOL 65 SEPTEMBER 2006
Table 2-Enzynlatic profiles using API zym system of LAB isolated from Chhu
(nanomotes) of LAB
'CUG3: RI CURl: Cl CUG2: R2 KCKI: RI KCK2: R4 KCYI:RI
Control (without enzyme)
Phosphatase alkaline
Esterase(C4)
Esteraselipase (C8)
Lipase (C14)
Leucine arylarnidase
Valine arylamidase
Cystine arylamidase
Trypsin
Chymotrypsin
Phoshatase acid
Napthol-AS- BI-phosphohydrolase
a-Galactosidase
f3-Galactosidase
f3-Glucuronidase
a-Glucosidase
f3-Glucosidase
N-Acetyl-f3-glucosarninidasc
u-Mannosidase
a-Fucosidase
o
o
5
5
o

5
o
o
o
5
10
10

o


o
o
o
o
o
5
10
o

30
30
o
o

20
o
10
o
5
5
o
o
o
CUGI: R2
o
o
5
o
o

5
5
o
o
5
10
30

o


o
o
o
o

5
5
o
30
o
o
o
o

30
10
10
o
10
10
o
o
o
o
o
5
o
o

5
5
o
o
5
10
30

o


o
o
o
o
o
5
5
o

5
5
o
o
10
5
30

o


o
o
o
o

5
5
o
30
o
o
o
o

20
10
20
o
10
10
o
o
o
Data represent the means of duplicate set
Table 3--Effect of LAB strains isolated from Chhu on coagulation and acidification of milk, and the degree of hydrophobicity
Product LAB strain Coagulation, 30" C
U
Coagulation; 37" Ch Hydrophobicity,
Control
Cow- Chhu
Yak-Chhu
Sterile skim milk
CUG3: RI
CURI:CI
CUGI: R2
CUG2: R2
KCKI: RI
KCK2: R4
KCYI: RI
- (6.8)
+ (5.0)
+ (4.5)
+(5.2)
- (5.4)
+ (4.6)
+ (4.5)
- (6.0)
- (6.8)
+ (5.4)
+(4.5)
+(5.1)
- (5.7)
+(5.0)
+ (5.1)
- (5.8)
75.3
97.2
96.3
79.2
85.5
82.1
86.0
Numbers in brackets represent the pH values; Data represent the means of triplicate sets.
occurred between 20-38 h; "coagulation occurred between 17-36 h.
milk by LAB strains reveals their potential as starters
or adjunct cultures in the production of Chhu.
Some strains of LAB that showed antagonistic
activities were not found to produce any bacteriocin
against indicator strains in the applied method. None
of the strains produced biogenic arnines in the applied
method. This is a good indication of their
acceptability in the possible development of starter
cultures. The production of biogenic amines by LAB
to be selected as starter cultures is not a desirable
property!". All strains of LAB showed a high degree
of hydrophobicity (>750/0), among which Lactococcus
lactis subsp. cremoris CURl:C1, isolated from
CC, showed highest percentage of hydrophobicity
(Table 3). Bacterial adherence to hydrocarbons, such
as hexadecane, proved to be a simple method to
determine cell surface hydrophobicity'". High degree
of hydrophobicity by LAB isolated frorn Chhu
probably indicates the potential of adhesion to gut
epithelial cells of human intestine, suggesting a
possible probiotic character", provided these strains
are consumed in a viable state.
DEWAN & TAMANG: MICROBIOLOGY OF CHHU
Table 4--Proxirnate composition of raw milk and fermented milk product (Chhu)
751
Parameter
pH
Turntable acidity as lactic acid)
Moisture, 0/0
DM
Fat, DM
Protein. DM
Carbohydrate, DM
Minerals, mg/ I 00 g DM
Calcium
Magnesium
Manganese
Iron
Zinc
Raw milk
6.7 0.09
0.15 0.01
87.0 2.12
5.5 0.76
30.8 4.37
28.0 0.41
35.8 2.78
103.8 1.45
39.7 0.85
1.2 0.1
1.7 0.1
57.7 0.75
Chhu
4.3 0.25
0.54 0.02
75.4 3.51
5.6 0.25
6.1 1.32
58.4 1.35
30.0 1.25
111.0 0.4
64.3 0.65
3.1 0.25
4.5 0.3
87.6 1.1
Raw milk
6.6 0.16
0.16 0.02
84:2 3.12
5.8 0.25
61.7 1.31
26.2 0.65
6.3 2.2
76.9 0.65
34.7 0.85
1.0 0.12
1.0 0.12
49.1 1.34
Chhu
4.8 0.28
0.45 0.02
70.2 4.12
5.9 0.67
11.2 0.92
61.8 2.14
21.1 2.07
89.0 0.73
50.1 1.24
1.5 0.25
2.70.12
56.6 0.68
Data renresent the means
Proximate composition (Table 4) of raw milk and
Chhu shows that Chhu was acidic (pH 4.3-4.8) in
nature, due to lactic acid fermentation. Like in typical
cheese products, high contents of protein and mineral
were observed in Chhu.
Conclusions
Indigenous knowledge of the people of Sikkirn for
production of fermented milks is sparse outside the
region. Chhu is an important dairy food in local diet,
which has not been documented yet. This study
showed that strains of LAB play an important role in
fermentation processes by their functional properties
related to partly wide enzyme spectrum, antagonistic
activities (though bacteriocin production was not
detected in the applied methods), coagulation and
acidification ability, probiotic properties (adherence
potential indicated by a high degree of
hydrophobcity), and even non-producers of biogenic
amines. Some of these LAB strains possess protective
and functional properties, which render them
interesting candidates for use as starter culture for
controlled and optirnized production of fermented
milk products.
Acknowledgements
Authors are grateful to Department of
Biotechnology, Ministry of Science and Technology,
Govt of India, for financial support.
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