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Food Biotechnology, 24:227247, 2010

Copyright Taylor & Francis Group, LLC

ISSN: 0890-5436 print

DOI: 10.1080/08905436.2010.507133

LFBT0890-54361532-4249Food Biotechnology,Vol. 24,No. 3, Jul2010: pp. 00Food Biotechnology

Microbiology and NutritionalValue of Selroti, an EthnicFermented Cereal Foodof the HimalayasMicrobiology of SelrotiH.Yonzanand J.P.Tamang

Hannah Yonzan and Jyoti Prakash Tamang

Food Microbiology Laboratory, Sikkim Government College, Sikkim University, Sikkim,India

Selroti is an ethnic fermented rice food of the Himalayas. A total of 125 samples of

selroti batters were collected from different villages and markets of the Himalayas. The

microbial population of selroti batters showed that lactic acid bacteria (LAB) were

present in viable numbers above 108 cfu/g, followed by yeasts at 105 cfu/g. LAB

Leuconostoc mesenteroides, Enterococcus faecium, Pediococcus pentosaceus andLactoba-

cillus curvatus and yeastsSaccharomyces cerevisiae,Saccharomyces kluyveri,Debaryo-

myces hansenii, Pichia burtonii, and Zygosaccharomyces rouxii were identified. The

most prevalent LAB and yeasts in selroti batters wereLeuc. mesenteroids (42.9%) and

S. cerevisiae (35.6%). Molds and pathogenic bacteria were not detected. It was observed

that seasons affect the development and prevalence of microorganisms in the fermented

batters. LAB and yeast strains were screened for their acidifying and coagulating capac-

ity, and it was found that most of the LAB strains acidified with lowering of pH up to

4.3. These strains showed a wide spectrum of enzymatic profiles in commercial API-zym

kits. All strains of LAB showed antimicrobial activities under the applied condition.

The nutritional value of fermented batters was found to be increased. This is the first

report on selroti concerning its microbiology and nutritional value.

Key Words: fermented food; LAB;Selroti; yeasts

INTRODUCTION

Cereals and their products are staple foods for billions of people throughout

the world. A global interest in rice and its fermented products is increasing

due to their easily available caloric value, unique quality characteristics, and

high acceptability. Cereals are fermented either to produce alcoholic beverages

and drink or to prepare varieties of baked products and staple nonalcoholic


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228 H. Yonzan and J.P. Tamang

foods (Sugihara, 1985). Nonalcoholic fermented cereal foods are mostly pre-

pared in the form of breads, loafs, confectionery, and gruels (Oyewole, 1997) as

well as complementary foods for infants and young children in Africa (Nout,

1991). Several fermented cereal products have been well investigated,

including sourdough of America, Australia, and Europe (Brandt, 2007); idli

of India (Steinkraus et al., 1967; Soni and Sandhu, 1991); dosa of India (Soni

et al., 1985);puto of Southeast Asia (Kelly et al., 1995); masa of South Africa

(Efiuvwevwere and Ezeama, 1996); maw or ogi of Benin (Onyekwere et al.,

1989; Hounhouigan et al., 1993a); kisra of Sudan (Mohammed et al., 1991);

ben-saalga of Burkino Faso (Tou et al., 2007); kenkey of Ghana (Nche et al.,

1994); togwa of Tanzania (Mugula et al., 2003); and tarhana of Turkey (Erbaset al., 2006).

Indigenous varieties of cereal crops are cultivated in the Himalayas

depending upon the agro-climatic conditions. These crops include rice, maize,

finger millet, wheat, barley, buckwheat, sorghum, and pearl millet. Rice and

maize are eaten mostly in the Eastern Himalayas; wheat is eaten as a staple

food in the Western Himalayas; and barley and finger millets are commonly

eaten in high mountain areas of the Himalayas (Tamang, 2010). Cereals are

mostly used as nonfermented staple foods and for the production of alcoholic

beverages in the Himalayas (Thapa and Tamang, 2004).Bhat, or cooked rice,

is a staple food in the Eastern Himalayas whereas chapatti or roti made from

wheat flour is common in the Western Himalayas.

The Nepali people of the Himalayan regions of India, Nepal, and Bhutan

prepare a fermented cereal food called selroti. It is a popular fermented rice

product that is ring shaped, spongy, pretzel-like, and deep-fried food (Fig. 1).

Selroti is consumed in religious festivals and special occasions (Yonzan and


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Microbiology ofSelroti 229

Tamang, 2009). During preparation, rice is soaked in cold water for 68 h.

Soaked rice is pounded into rice flour and mixed thoroughly with about 25%

refined wheat flour, 25% sugar, 10% butter or fresh cream, and 2.5% spices/

condiments containing large cardamom, cloves, coconut, fennel, nutmeg, and

cinnamon. Milk is added, kneaded into soft dough, and finally made into batter

with easy flow. Batter is left to ferment naturally at ambient temperature

(2028C) for 24 h during the summer and at 1018C for 68 h during winter.

The fermented batter is squeezed deposited as continuous ring onto hot edible

oil, fried until golden brown, and drained out from hot oil by a poker or spatula.

Selroti is served as confectionary bread. It can be stored at room temperature

for two weeks. The preparation ofselroti is an art of traditional technology,which is passed from mother to daughter. Women prepare it and men help

them in pounding the soaked rice. It is also sold in canteens, local food stalls,

and restaurants. To the best of our knowledge, no microbiological and

biochemical aspects of selroti of the Himalayas have been investigated. The

present study focused on the microbiology and nutritional value ofselroti with

the long-term goal of developing good starter culture technology for this

fermented food.

MATERIALS AND METHODS

Collection of Samples

A total of 78 samples of home-made selroti batters were collected directly

from villages located in Darjeeling hills and Sikkim in India. Similarly, 36

market samples of selroti batters were collected from different restaurants,

local food stalls, and canteens located at Gangtok in Sikkim, and 11 lab-made

samples ofselroti batters were collected aseptically in sterile bottles.

Microbial Analysis

Ten g of sample was suspended in 90 mL of 0.85% (w/v) sterile physiological

saline and homogenized in a stomacher lab-blender 400 (Seward, UK) for

1 min. Decimal dilution series were prepared in sterile diluents, and diluted

suspension of sample was mixed with the molten media and poured into

plates. Lactic acid bacteria (LAB) were selectively isolated on MRS agar

(M641, HiMedia, India) plates supplemented with 1% CaCO3 and incubated

under anaerobic condition in an Anaerobic Gas-Pack system (LE002, HiMedia,

India) at 30C for 3 d. Aerobic mesophilic counts (AMC) were determined

using plate count agar (M091A, HiMedia, India) which was incubated at 30Cfor 48 h. Plates of potato dextrose agar (M096, HiMedia, India) and yeast

e tract malt e tract agar (M424 HiMedia India) for e amination of molds


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of colony forming units (cfu) of microorganism was counted for determination

of total microbial population of the sample.

Samples were tested for presence ofBacillus cereus using selectiveBacillus

cereus agar base (M833, HiMedia, India),Staphylococcus aureus using Baird

Parker agar base (M043, HiMedia, India) and enterobacteriaceae using Violet

Red Bile Glucose agar (M581, HiMedia, India) following the methods

described by Han et al. (2001). Salmonella-Shigella Agar (M108, HiMedia,

India) was used for the detection ofSalmonella and Shigella and Listeria

identification agar base (M1064, HiMedia, India) with Listeria selective sup-

plement (FD 061, HiMedia, India) for Listeria in the samples following the

standard method of Metaxopolous et al. (2001).

Characterization and Identification

Cell morphology of all bacterial isolates and their motility were deter-

mined using a phase contrast microscope (CH3-BH-PC, Olympus, Japan).

Bacterial isolates were Gram-stained and tested for catalase production by

placing a drop of 10% hydrogen peroxide solution on isolates and were prelim-

inarily identified on the basis of carbon dioxide production from glucose,

ammonia production from arginine, growth at different temperatures (10C,

15C, 45C), the ability to grow in different concentrations of sodium chloride(6.5%, 10%, 18%), and pH (3.9, 9.6) in MRS broth (M369, HiMedia, India),

following the methods of Schillinger and Lcke (1987). Dextran production

was tested by growing the culture on the 5% (w/v) sucrose agar (Garvie, 1984)

and observed for mucoid appearance on the agar plates (Kelly et al., 1995).

The configuration of lactic acid produced from glucose was determined enzy-

matically using commercial D-lactate and L-lactate dehydrogenase test kits

(Boehringer-Mannheim GmbH, Germany). Carbohydrate fermentation pat-

terns of LAB isolates (grown on MRS agar at 30C for 48 h) were determined

using API 50 CHL and API 20 STREP test strips (bioMrieux, France) accord-

ing to the manufacturers instructions. The results were read by referring tothe manufacturers interpretation using the APILAB PLUS database identifi-

cation software (bioMrieux, France). Based on physiological tests and API

sugar profiles, LAB isolates were identified following the Bergeys Manual of

Systematic Bacteriology (Sneath et al., 1986). Characterization and identification

of yeasts were carried out following the method of Kurtzman and Fell (1998).

Effect of Seasonal Variation on Microbial Population

The effect of seasonal variation on microbial population of selroti batters

prepared during summer and winter was studied. Samples were collected dur-ing summer (May to July) and winter (December to February) from different

t t f d t ll t d t t d l t d i D j li hill


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Acidification and CoagulationAcidification and coagulation properties were assayed by inoculating 10%

skim milk (RM1254, HiMedia, India) with LAB strains. Observation was

made for commencement of clotting, and the pH was measured after 72 h of

incubation at 300C (Olasupo et al., 2001).

Enzymatic Activities

The enzymatic activities of LAB strains were assayed using the commer-

cial API-zym (bioMrieux, France) galleries. Cultures were grown on MRS

agar for 48 h and were centrifuged, supernatant discarded, and the precipi-tates (cells) mixed aseptically with 2 mL sterile normal saline, which was used

to prepare suspension of 107 cells/mL. The strip was unpacked, and 2 drops of

cell suspensions were inoculated in each cupules of the strip containing ready-

made enzyme substrates and incubated at 30C for 6 h. After incubation,

1 drop of ready-made zym-A and zym-B reagents was added and observed for

color development based on the manufacturers color chart. A value ranging

from 05 was assigned, corresponding to the colors developed: 0 corresponds

to a negative reaction, 5 (= 40 nanomoles) to a reaction of maximum intensity,

and values 4, 3, 2, and 1 were intermediate reactions corresponding to 30, 20,

10 and 5 nanomoles, respectively.

Antimicrobial Activity

Lactic acid bacteria isolated from selroti batters were tested for antimicro-

bial activity by the agar spot method (Schillinger and Lcke, 1989) against

several reference bacteria. References bacteria were Listeria innocua DSM

20649,L. monocytogenes DSM 20600,Bacillus cereus CCM 2010,Staphylococcus

aureus S1,Pseudomonas aeruginosa BFE 162,Enterobacter agglomerans BFE

154,E. cloacae BFE 282, andKlebsiella pneumoniae subsp.pneumoniae BFE

147. Originally, these reference strains were obtained from DSM (Deutsche

Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany),

CCM (Czechoslovak Collection of Microorganisms, Brno, Czechoslovakia),

BFE (Institute of Hygiene and Toxicology, Karlsruhe, Germany), and FMR

(Food Microbiology Laboratory, Sikkim Government College, Gangtok, India).

Listeria monocytogenes DSM 20600,Staphylococcus aureus S1,Bacillus cereus

CCM 2010, and Klebsiella pneumoniae subsp. pneumoniae BFE 147 were

propagated in standard nutrient agar. The cultures were maintained as frozen

stocks at -20C in 15% glycerol.

Cell-free neutralized supernatants of LAB isolates were screened for bac-teriocin activity by the agar spot test method (Uhlman et al., 1992) using the

bacteriocin screening medium (MRS agar supplemented with 0 2% glucose) as


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broth were centrifuged followed by filtration of the supernatant through a 0.2

m

pore-size cellulose acetate filter. The cell-free supernatant was adjusted to pH

6.5 by addition of 1N NaOH and stored frozen until tested. The cell free

extracts were spotted onto soft MRS agar (containing 0.7% agar) plates, inocu-

lated with indicator strains, were incubated at 30oC for 24 h, and subse-

quently examined for zone of inhibition.

Analysis of Nutritional Value

Ten g of sample were mixed with 20 mL carbon dioxide-free distilled water

in a blender for 1 min and the pH of the slurry was determined directly(AOAC, 1990) using a digital pH meter (Model 361, Systronics, India) cali-

brated with standard buffer solutions (Merck, Germany). Titratable acidity of

sample was calculated by titrating the filtrates of a well blended 10 g sample

in 90 mL carbon dioxide-free distilled water with 0.1 N sodium hydroxide to

end point of phenolphthalein (0.1% w/v in 95% ethanol) (AOAC, 1990).

Moisture content was determined by weight loss of accurately weighed 1 g

of sample (in triplicate) after heating at 135oC for 2 h. Reducing sugar content

of the sample was determined by the colorimetric method of Somogyi (1945)

using glucose as standard solution. Total sugar of the sample was estimated

by determining reducing sugar in hydrolysed sample with HCl (AOAC, 1990).Ash content was measured by heating the sample at 550C until the difference

between two successive weighing was 1mg (AOAC, 1990). Water-soluble

nitrogen and trichloroacetic acid (TCA)-soluble nitrogen of the samples were

determined as described by Tamang and Nikkuni (1996). Protein content was

determined by multiplying total nitrogen, estimated by standard Kjeldahl

method, by 6.25 (AOAC, 1990). Fat content was determined by ether extrac-

tion using glass soxhlet (AOAC, 1990). Carbohydrate content was estimated

by difference: 100 (% protein + % fat + % ash) (Standal, 1963). Calcium,

sodium, and potassium were estimated in flame-photometer (Model CL 361,

Elico, India). Energy value of a sample was estimated as the method described

by Indrayan et al. (2005).

RESULTS

Microbial Population

A total of 125 samples ofselroti batters were analyzed for microbiological

populations (Table 1). In home-made samples ofselroti batters, the microbial

populations of LAB and yeasts were 104 to 108 cfu/g and 104 to 105 cfu/g,respectively. The average count of LAB and yeasts in market samples of

f t d b tt 108 f / d 105 f / i l h h


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and 105 cfu/g, respectively. Mycelial fungi, Bacillus cereus, Listeria sp.,

Salmonella sp., and Shigella sp. were not detected in any sample of fer-

mented batters. Counts of enterobacteriaceae andStaphylococcus aureus were

detected below 102 cfu/g. No bacterial contaminants were detected in any sample

of lab-made selroti batters.

LAB Strains

A total of 167 bacterial isolates were isolated from selroti batters collected

from different sources. All isolates were purified in MRS broth, and their cell

morphology and preliminary taxonomical tests were performed. All bacterial

isolates were considered lactic acid bacteria due to their growth in anaerobic

agar, formation of clear zones around colony in MRS agar plates supple-

mented with calcium carbonate indicating the hydrolysis of carbonates due to

acid production by LAB, and were Gram-positive, catalase-negative, nonmo-

tile, and nonsporing. A grouping of all LAB isolates was based on cell morphol-

ogy, gas production from glucose and arginine hydrolysis (Table 2). The

representative strains of LAB were selected randomly from each grouped

strains having similar morphology, the ability to produce gas from glucose and

hydrolyse arginine, and isolated from the respective samples. All cocci forming

tetrads were presumptively grouped as pediococci. Further differentiation of

all tetrad forming strains was performed by using the key proposed by Simp-

son and Taguchi (1995) based on the ability to grow at maximum pH 8.5 and

minimum pH 4.2, at 50C and in the presence of 10% NaCl (data not shown).On the basis of these tests, tetrad strains L9:B2, S4:B2, S5:B2, BG2:B2,

L1:B3 L3:B3 S2:B3 BP:B3 BG3:B2 BN1:B3 BN2:B3 BA1:B3 were iden-

Table 1: Microbial populations of selrotibatters.

Source

Log cfu/g sample

LAB Yeast AMCEnterobacteria

ceaeStaphylococcus

aureus

Home-made selroti(n = 78)

6.6 0.2 5.1 0.2 6.8 0.2 2.3 0.4 1.1 0.1

Market-made selrotibatter (n = 36)

8.1 0.3 5.8 0.2 8.3 0.2 2.1 0.1 1.3 0.3

Lab-made selrotibattera (n = 11)

7.6 0.7 5.3 0.3 7.9 0.4 0 0

aselrotiprepared at Laboratory following the traditional method.n, number of samples collected.Data represents the means ( SD) of number of samples.LAB, lactic acid bacteria; AMC, aerobic mesophilic count.Molds, Bacillus cereus, Listeriasp. Salmonellasp. and Shigellasp. were not detected in anysample.


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e2:Gr

oupingofrepresentativestra

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ainsof

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9/22


API identification profile also confirmed the identity of tetrad strains as

P. pentosaceus. All homofermentative rod strains isolated from selroti batters

were curved, bean-shaped rods with rounded ends, in pairs, and short chains

and closed rings of usually four cells or horse-shoe forms were frequently

observed. These strains grew well at 15C and produced DL lactate from

glucose. They were able to ferment ribose, trahalose, mannose, esculin,

salicin, cellobiose, and maltose as shown in the API test and were identified

asLactobacillus curvatus. All coccoid strains isolated from selroti batters pro-

duced D (-) lactate from glucose, were arginine-negative, showed the typical

leuconostoc-like ovoid cells, and produced dextran when grown on 5% sucrose

agar. However, they fermented sucrose, galactose, maltose, mannose, andxylose. Sugar fermentation profiles using API confirmed their identity as

Leuconostoc mesenteroides. Cocci strains (L1:B2, L3:B2, S1:B3, L6:B4, S5:B6,

BS1:B2, BP:B2, L10:B3, BG3:B3, S1:B4, S4:B4) were nongas producer,

arginine-positive, grew well in 6.5% NaCl, and at 45oC. They were identified

asEnterococcus faecium.

Yeast Strains

A total of 141 yeast isolates were isolated from selroti batters collected

from different sources. The representative strains of yeast were selected ran-domly from each grouped strains having similar colony appearance, cell

shape, type of mycelia, and ascopsore for detailed identification (Table 3).

Sugar fermentation and assimilation tests of randomly selected representa-

tive strains of yeasts were carried out. Following the taxonomical keys of

Kreger-van Rij (1984) and Kurtzman and Fell (1998), strains BG1:Y1,

BA1:Y1, BG3:Y1, S1:Y1, L1:Y1 had dusty, dry, and powdery surfaced colonies

fringed with many strands of mycelia when grown on agar plates. They

formed expanding septate hyphae with conidia borne on denticles. There were

1 to 4 hat-shaped ascospores per ascus. All of them fermented glucose, galac-

tose, maltose, raffinose, and sucrose. They were able to grow in 10% NaCl and

5% glucose in yeast nitrogen base. As a preliminary step they were identified

asPichia burtonii. Yeast strains BG1:Y2, BA1:Y2, S2:Y2, BA2:Y5, S1:Y5 and

L1:Y7 had smooth surfaced colonies, showing globose ascospores and fermented

vigorously, and as a preliminary step identified as Saccharomyces cerevisiae.

Yeast strains BG1:Y3, S1:Y3, S3:Y3 and L3:Y3 had smooth surfaced colonies

fringed with pseudohyphae, showing globose ascospores and fermented

sucrose. They were identified as Saccharomyces kluyveri. Yeast strains

BG1:Y4, BR1:Y4, S4:Y4, S5:Y4, and L1:Y4 showed smooth surfaced colonies

with spheroidal ascospores and fermented glucose weakly. The preliminaryidentification indicated that it isDebaryomyces hansenii. Yeast strains S1:Y6

and L9:Y6 showed smooth surfaced colonies with 14 globose ascospores and


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11/22


containing 10% glucose, 1% tryptone, 1% yeast extract, 2% agar, and 1%

acetic acid within 3 d. These strains as a preliminary step identified as

Zygosaccharomyces rouxii.

Prevalence of Microorganisms

The most dominant LAB in all samples of selroti batters were Leuc.

mesenteroides at 42.9%, followed byP. pentosaceus (23.8%),E. faecium (20.4%),

andL. curvatus (13.0%) out of 167 strains of LAB (Fig. 2). The most dominant

yeast recovered in all samples ofselroti batters wereS. cerevisiae, which repre-

sented 35.6% of all yeasts, followed byD. hansenii (17.6%),P. burtonii (17.1%),

Z. rouxii (16.3%), andS. kluyveri (13.4%) out of 141 isolates of yeasts (Fig. 3).

Figure 2: Graphic representation of average prevalence of functional LAB in selrotibatters.

42.9

23.8

20.4

13

0

10

20

30

40

50

%of

Prevalence

Types of LAB

Leuconostocmesenteroides

Pediococcuspentosaceus

Enterococcus faecium

Lactobacillus curvatus

35.6

17.6 17.1 16.3

13.4

0

10

20

30

40

%o

fPrevalance

Types of Yeasts

Saccharomycescerevisiae

Debaryomyceshansenii

Pichia burtonii

Zygosaccharomycesrouxii

Saccharomyceskluyveri


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Effect of Seasonal Variation on Microbial LoadThe effect of seasonal variation on microbial load of selroti batters pre-

pared during summer and winter seasons was studied. A total of 36 samples

each were collected during summer and winter from different restaurants,

food stalls, canteens, and street vendors located in Darjeeling hills and

Sikkim. During the summer, it was observed that the microbial load of LAB

and yeast was found 108 cfu/g and 104-105 cfu/g, respectively. During winter,

the microbial populations were about 107 cfu/g, slightly lower than that

observed during summer. However, yeasts population increased up to 106 cfu/g

during winter (data not shown). The average maximum temperature at

Gangtok during summer is 22.2C and the temperature in winter is 13.8C.The average pH of the samples during summer and winter was 4.7 and 5.0,

respectively. The titratable acidity was 0.11 and 0.09 during summer and winter,

respectively.

Effect of Acidification and Coagulation

TheE. faecium strains BP:B2 and S4:B4 showed the lowest acidification

value of pH 4.3 among all the tested strains of LAB, followed by E. faecium

strains S5:B6, BS1:B2, L10:B3, and S1:B4, dropping the pH to 4.4. About

63.6% of LAB strains caused coagulation of milk at 30C with a significant drop

in pH.L. curvatus, Leuc. Mesenteroides, andE. faecium coagulated skim milk

within 2430 h at 30C. The fastest coagulation time of 2428 h was observed in

several strains ofLeuc. mesenteroides. However, strains ofP. pentosaceus did

not coagulate skim milk.

Among yeasts, S. cerevisiae strain S2:Y2 showed the lowest acidification

value of pH 5.6, followed byS. cerevisiae strains BA1:Y2, BA2:Y5, and S1:Y5

andD. hansenii BR1:Y4, dropping the pH to 5.7.S. cerevisiae andD. hansenii

coagulated skin milk. The coagulation time ranged from 3440 h at 28 oC.

The most rapid coagulation time of 3436 h was observed in many strains ofS. cerevisiae. However, none of the strains ofP. burtonii, S. kluyveri, and

Z. rouxii showed coagulating abilities in the applied method.

Enzymatic Activities

Enzymatic activities of LAB and yeast strains were assayed using the

commercial API-zym (bioMrieux, France) galleries (Tables 4 and 5). The LAB

strains showed relatively weak esterase (C4), moderate phosphatase, and

strong arylamidase activities. However,E. faecium strains BS1:B2 and S1:B3

showed moderate proteinase activity, whereas, phosphohydrolase activity wasshown by all strains tested.Lb. curvatus strains BP:B1 and BS1:B1 showed the

highest activity (>40 nanomoles) of a glucosidase Leuc mesenteroides strains


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strains showed the highest b-glucosaminidase activities among the LAB

strains. Yeast strains showed relatively weak esterase (C4) and weak to

strong arylamidase and strong phosphatase activities (Table 5). However, the

strains showed no detectable proteinase activity. Acid phosphatase activity

was shown by all strains tested, among which >40 nanomole activities was

shown by D. hansenii, P. burtonii, and Z. rouxii strains. Phosphohydrolase

activity was also shown by all strains tested.

Screening of Bacteriocin Activities

The anitimicrobial activities of LAB strains were tested against Bacillus

cereus CCM 2010,Klebsiella pneumoniae subsp.pneumoniae BFE 147,Listeria

monocytogenes DSM 20600, and Staphylococcus aureus S1. Most of the LAB

strains showed the clear inhibition zones in agar-spot plates against these

bacteria (data not shown). Cell-free supernatant extract of LAB strains were

tested for bacteriocin assay. None of the LAB strains produced bacteriocin

under the applied conditions.

Nutritional Value

The proximate composition of unfermented rice and wheat flour and sam

Table 4: Enzymatic activities of LAB strains from selrotibatters using API-zym.

Enzyme

LAB strains (Activity in nanomolesa)

BP:B1 S5:B1 BS1:B1 S6:B1 BS1:B2 S1:B3 BG2:B2 S4:B2

Esterase lipase (C8) 5 5 0 0 10 5 5 5Lipase (C14) 5 5 0 0 0 0 5 5Leucine arylamidase 40 30 20 10 5 10 40 40Valine arylamidase 40 30 0 0 0 0 30 30Cystine arylamidase 30 30 5 10 0 0 20 20a-chymotrypsin 0 0 0 0 30 20 0 0Acid phosphatase 10 10 10 10 5 5 10 10

Napthol-AS-BI-phosphohydrolase 10 10 10 20 10 20 30 30a-galactosidase 5 5 40 40 0 0 0 0-galactosidase 0 0 40 40 5 5 0 0

a-glucosidase 40 40 0 0 0 0 0 0-glucosidase 40 40 5 5 40 30 20 30

N-acetyl-b-glucosaminidase

30 20 0 0 10 10 40 40

Data represent the means of 3 sets of experiment.a0, no enzyme activity; 5, 10, 20, 30, >40 indicates nanomoles of hydrolysed substrate after6 h of incubation at 30o C.Alkaline phosphatase, esterase (C4), trypsin, b-glucuronidase, a-mannosidase and a-fucosidasewere not hydrolysed by any LAB strain.BP:B1, L. curvatus; S5:B1, L. curvatus; BS1:B1, Leuc. mesenteroids; S6:B1, Leuc. mesenteroids;

BS1:B2, E. faecium; S1:B3, E. faecium; BG2:B2, P. pentosaceus; S4:B2, P. pentosaceus.


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e5:En

zymaticactivitiesofyeaststra

insfroms

elrotib

attersusingA

PI-zym.

me

Yeatsstra

ins(Activ

ity

innanomolesa)

BR1:Y

4

S4

:Y4

BG1:Y

1

S1:Y

1

BA1:Y

2

S2:Y

2

S3:Y

3

BG1:Y

3

S1:Y

6

L9:Y6

ineph

osphatase

30

20

20

20

5

5

5

10

0

0

ase(C

4)

5

5

5

5

5

5

5

5

5

5

aselip

ase(C8)

5

5

10

10

10

10

5

5

10

20

cinearylamidase

5

5

30

30

30

40

20

20

20

20

nearylamidase

0

0

0

0

20

20

5

5

10

5

phosp

hatase

40

40

40

40

30

30

20

30

40

40

thol-AS-BI-

ospho

hydrolase

20

20

30

30

20

30

30

30

30

20

cosidase

5

5

20

20

30

40

40

40

0

0

cosidase

0

0

20

20

10

30

30

20

0

0

cetyl-b-

ucosam

inidase

5

5

5

5

0

5

0

0

5

0

cosidase

5

10

0

0

5

5

0

0

0

0

representthemeansof3setsofexperiment.

oenzym

eactivity;5,10,20,30,>40indic

atesnanomolesofhydrolysedsu

bstrateafter6hofincubationat30oC.

e

(C14

),cystine

arylamidase,trypsin,

a-chymotrypsin,a-galactosidase

,b-galactosidase,

b-glucuronida

se

and

a-mannosidase

weren

ot

olysedbyanyyeaststrain.

4,

D.h

ansenii;S4:Y1,

D.

hansenii;BG1:Y

1,

P.

burtonii;S1:Y1,

P.

burtonii;BA1:Y2,

S.

cerevisiae;S2:Y2,

S.

ce

revisiae;S3:Y3,

S.

kluyveri;BG1:Y

3,

yveri;S1:Y6,

Z.

rouxii;L9:Y6,

Z.

rouxii.


15/22


Food value and limited mineral contents (calcium, sodium and potassium)

were also determined. The content of moisture, reducing sugar, total sugar,

fat, and carbohydrate in selroti batters increased compared to the raw materials.

There was a marked increase in water-soluble nitrogen and TCA-soluble

nitrogen in products over the raw materials. The energy value of fermented

batters increased slightly more than the unfermented raw materials (Table 6).

Contents of sodium and calcium increased in fermented products.

DISCUSSION

The microbial population of selroti batters collected from different sources

revealed that LAB, comprising lactobacilli, pediococci, leuconostocs, and

enterococci, were the predominant microorganisms present in viable numbers

above 108 cfu/g, followed by yeasts at 105 cfu/g. Taxonomically diverse species

of LAB have been identified from selroti batters, which included Leuc.

mesenteroides, E. faecium, P. pentosaceus, andL. curvatus.Leuc. mesenteroides

has been reported in several fermented cereal foods such as idli of India

(Mukherjee et al., 1965), enjera of Ethiopia (Oyewole, 1997), puto of thePhilippines (Kelly et al., 1995), and maw of Togo and Benin (Hounhouigan

t l 1993 ) E f i t l i k f i f d b

Table 6: Proximate composition of selroti.

Parameter

Raw materialsaFermented

product

Rice (n = 6)Wheat flour

(n = 6)selrotibatter

(n= 46)

pH 5.5 0.1 5.9 0.1 5.8 0.4Titratable acidity % (as lactic acid) 0.09 0.01 0.1 0.01 0.08 0.01Moisture % 16.3 0.4 18.4 0.7 42.5 4.6Reducing sugar % 0.01 0.01 0.02 0.01 2.1 0.5Total sugar % 63.8 0.9 58.4 1.2 69.2 4.4

Ash (% DM) 0.7 0.06 0.5 0.07 0.8 0.08Fat (% DM) 1.0 0.01 0.9 0.01 2.7 0.3Water-soluble nitrogen (% DM) 0.016 0.01 0.056 0.01 0.06 0.02TCA-soluble nitrogen (% DM) 0.0016 0.001 0.0017 0.003 0.004 0.002Protein (% DM) 8.3 0.01 11.0 0.5 5.7 0.5Carbohydrate (% DM) 90.0 1.0 87.6 0.9 91.3 0.6Sodium (mg/100 g) 5.9 0.7 5.9 0.5 8.9 0.6Potassium (mg/100 g) 47.4 1.1 117.5 2.5 29.7 1.1Calcium (mg/100 g) 9.4 0.5 20.8 0.2 23.8 1.6Energy value (Kcal/100g DM) 402.2 0.4 402.5 0.5 410.3 0.5

n, total number of samples (n) collected from each source is given in parenthesis.Data represent the means ( SD) of triplicate of each sample.DM, dry matter. TCA, trichloro-acetic acid.aRaw materials purchased from Gangtok.


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E. faecalis (Franz et al., 2003). P. pentosaceous along with several species of

lactobacilli were reported as predominant LAB strains in Tanzanian togwa

(Mugula et al., 2003), in maw of Togo and Benin (Hounhouigan et al., 1993b),

and in kodo ko jaanr of India (Thapa and Tamang, 2004).L. curvatus has been

reported in maw, a fermented maize food in lesser percentage of prevalence

than other LAB (Hounhouigan et al., 1993b).

Yeasts strains S. cerevisiae, S. kluyveri, D. hansenii, P. burtonii, and

Z. rouxii were also isolated from selroti batters, which predominately co-exited

with LAB. Yeast fermentation serves several functions in sourdough produc-

tion as well as other fermented cereals (Hammes and Ganzle, 1998; Tamang

and Fleet, 2009). Gas production causes expansion and leavening of thedough, ultimately affecting the texture, density, and volume of the bread

(Hammes et al., 2005). S. cerevisiae is the principal yeast of most bread

fermentations (Jenson, 1998).S. kluyveri has been reported in nan, a leaved

bread of North India (Batra, 1986). D. hansenii has been isolated from idli

along with several other yeasts (Soni and Sandhu, 1991).P. burtonii has been

reported in some Asian rice-based alcoholic starters such as loog-pang of

Thailand (Limtong et al., 2002) and marcha of Sikkim (Tsuyoshi et al., 2005).

However,P. burtonii produces visible, white, or chalky discoloration in sour-

dough (Legan and Voysey, 1991). Acid-tolerantZ. rouxii has not been reported

in fermented cereal-based foods, though it has been reported in many

fermented soybean foods of Asia which contribute aroma to the product (Aidoo

et al., 2006). Origin ofZ. rouxxii is usually from sugar, honey, and confectionery

(Kreger-van Rij, 1984). Probably recovery ofZ. rouxii in selroti batters was

likely due to their entry through sugars and honey, which are added during

selroti batter preparation to make it sweet. The most prevalent LAB and

yeasts in all samples ofselroti batters wereLeuc. mesenteroids andS. cerevisiae,

respectively, which were recovered in all samples analyzed as predominant

organisms. Predominance ofLeuc. mesenteroids andS. cerevisiae was common

in other fermented cereal-based foods (Steinkraus, 1996; Brandt, 2007).

Food-borne pathogens Bacillus cereus, Listeria sp., Salmonella sp. and

Shigella sp. were not detected in any sample of fermented batters of selroti

due to the slightly acidic nature of the products. High population (>108 cfu/g)

of LAB in selroti batters could restrict the growth of other organisms simply

by their physical occupation of available space and uptake of most readily

assimilative nutrients (Adams and Nicolaides, 1997). Lactic acid produced by

LAB may reduce pH to a level where pathogenic bacteria may be inhibited

(Tsai and Ingham, 1997; Adams and Nout, 2001). Another safety aspect of

selroti is deep frying prior to consumption. There has been no report of any

food poisoning or infectious disease infestation by consuming selroti.Acidification is an important technological property in relevance of selec-

tion for starter culture among the LAB (de Vuyst 2000) About 63 6% of LAB


17/22


strains shows their potential as starters or adjunct cultures in the production

of fermented products. Among yeasts strains, onlyS. cerevisiae andD. hansenii

showed acidification characters, though the decrease in pH was limited to 5.6.

The casein degradation initiated with milk clotting peptidases and proteinases,

which produce peptides and amino acids (Myra-Mkinen and Bigret, 1998).

The use of the API-zym technique has relevance for selection of strains as

potential starter cultures based on superior enzyme profiles, especially pepti-

dases and esterases, for accelerated maturation and flavor development in fer-

mented products (Tamang et al., 2000; Kostinek et al., 2005). The absence of

proteinases (trypsin) and the presence of strong peptidase (leucine-, valine-,

and cystine-arylamidase) activities produced by the predominant LAB strainsisolated from selroti batters are possible traits of desirable quality for their

use in production of typical flavor and aroma. High activity of phosphatase

by yeast strains showed their possible role in phytic acid degradation in

cereal-based fermented foods. Anti-nutritive factors such as phytic acids and

oligosaccharides are of particular significance in unbalanced cereal-based

diets (Fredrikson et al., 2002). Due to these nutritional consequences, the

degradation of anti-nutritive factors in food products by fermentation is desir-

able (Chavan and Kadam, 1989; Svanberg et al., 1993). The presence of high

activity of a-galactosidase by Leuc. mesenteroides probably indicated their

ability to hydrolyze oligosaccharides of raffinose family (Holzapfel, 2002).

It was observed that seasons affect the prevalence of microorganisms in

the fermented batters. During summer, the microbial load of LAB increased

due to a rise in temperature, which may accelerate fermentation rate; winter

was favorable for yeasts. Similar observation on effect of seasonal variation

was made during idli fermentation favoring the bacterial load (Soni et al.,

1986).

Moisture content in selroti batters was higher than that of raw materials

due to soaking prior to fermentation and to the addition of water and milk

during its preparation. There was a remarkable increase in water-soluble and

TCA-soluble nitrogen in selroti batters due to solubilization of proteins, indi-

cating its protein digestibility. Increase in free amino acids in tarhana has

been reported (Erbas et al., 2005). Food value of selroti batters are almost

same as reported in other fermented cereal foods such as idli (Soni and

Sandhu, 1989) and tarhana (Erbas et al., 2005).

CONCLUSION

Selroti is a unique fermented cereal food of the Nepali people in the Himalayas.

Lactic acid bacteria and yeasts co-exited as the predominant organisms inselroti, enhancing the functional properties as well as food value of the product.

Th i l t d i i t ib t t th d l t f th


18/22


Himalayas. Development of these microbial resources has benefits for devel-

opment of specific starter cultures and also use of unique microbial processing

for development of functional foods using cereals of the Himalayas as a funda-

mental resources to add value.

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