As a library, NLM provides access to scientific literature.

Inclusion in an NLM database does not imply

endorsement of, or agreement with, the contents by NLM or the National Institutes of Health.
Learn more: PMC Disclaimer | PMC Copyright Notice

J Food Sci Technol. 2018 Sep; 55(9): 3417–3426. PMCID: PMC6098797

Published online 2018 Jul 26. doi: 10.1007/s13197-018-3247-2 PMID: 30150800

Shelf life improvement of idli batter by addition of mustard essential oil as bio-preservative
Baburaj Regubalan and Laxmi Ananthanarayan

Abstract

Idli is one of the most popular naturally fermented breakfast food. In this study, essential oils have been screened for
their minimal inhibitory activity against selected lactic acid bacteria (LAB) and yeast strain associated with idli batter
fermentation and to identify the best potential bio preservative to preserve the idli batter. Mustard essential oil was
found to be the best bio-preservative which showed a biocidal effect at 80 ppm against LAB strains and at 40 ppm
against Candida versatilis. The efficacy of mustard essential oil incorporated in the idli batter at 0.1% (w/w) was eval‐
uated by measuring the titratable acidity, pH, viscosity, batter volume, microbial count of idli batter as well as sensory
parameters of idli prepared from preserved batter stored at 4 and 30 °C. Unfavorable changes in acidity, batter volume
and whey separation of idli batter containing mustard oil were significantly reduced, which resulted in a reduction of
sour taste and improved texture of idli. The growth of yeast and LAB was retarded evidenced by decreased microbial
counts than control batter, which delayed the deterioration of the batter under both storage conditions. The addition of
0.1% mustard essential oil in the optimally fermented idli batter extended its shelf life to 5 days when stored at 30 °C
and 30 days at 4 °C.

Electronic supplementary material

The online version of this article (10.1007/s13197-018-3247-2) contains supplementary material, which is available to
authorized users.

Keywords: Idli batter, MIC, Mustard essential oil, Shelf life

Introduction

“Idli” is a traditional steam cooked, fermented food which is known for its typical flavor, characteristic sour taste, soft,
spongy texture, high nutrient content, and easy digestibility. It is one of the major breakfast foods in India, Sri Lanka,
Malaysia and Singapore (Steinkraus 1995; Durgadevi and Shetty 2012; Shrivastava and Ananthanarayan 2015). Rice
(Oryzae sativa) and black gram dal (Phaseolus mungo) are the two main ingredients taken generally in the ratio of
3:1–4:1 for idli batter preparation, after which the batter is allowed to ferment spontaneously. LAB convert availableBack to Top
carbohydrates into organic acids viz. lactic acid which reduces the pH of the batter, favoring the growth of yeast
(Sridevi et al. 2010; Durgadevi and Shetty 2012). The indigenous bacteria reported in idli batter are Leuconostoc
mesenteroides, Lactobacillus delbrueckii, L. fermentum, L. lactis, L. brevis, and Pediococus cerevisiae; Additionally,
yeasts such as Candida glabarata, Debaryomyces hansenii, Geotrichum candidum, Saccharomyces cerevisiae,
Torulopsis holmii, Trichosporon pullulans, T. beigelli, Candida fragilola, C. kefyr, C. tropicalis, C. versatilis and
Hansenula anomala have also been found to play a major role in idli fermentation (Steinkraus 1995; Saravanan et al.
2015; Saravanan and Shetty 2016). The shelf life of the fermented idli batter is very short and it can be stored under re‐
frigerated conditions for about 5 days (Nisha et al. 2005).

Idli batter manufacturing industry is a very flourishing business in India with some manufacturers producing up to
55,000 kg of batter per day (Dhamija 2017), which is then distributed to retail outlets in smaller packages to be sold di‐
rectly to the consumers. A survey of locally prevalent idli batter distribution outlets also indicates that refrigeration of
idli batter (4–8 °C) is the most commonly practiced method used to extend the shelf life for 5–7 days by inhibiting the
microbial growth. The major problems faced in idli batter distribution are puffing and bursting of packets due to over
fermentation by wild indigenous microbes and improper storage conditions. Further, high acidity and whey separation
in the idli batter results in very sour tasting and hard textured cooked idlis (Nisha et al. 2005). There is thus a great
need to identify potential antimicrobial additives that can inactivate or inhibit the growth of LAB and yeast in the bat‐
ter system after an optimized period of fermentation (12 h) that will result in the extension of shelf life of idli batter.

Essential oils are aromatic oils obtained from plant sources; they are well known for their antimicrobial activity
(Kalemba and Kunicka 2003; Burt 2004; Ribeiro-santos et al. 2017) and hence they are useful as bio-preservatives.
Minimum Inhibitory Concentration (MIC) is commonly used to screen the potential antibacterial activity of sub‐
stances. MIC depends on many factors such as the method of extraction of essential oils, inoculum size, growth phase,
culture medium, pH of media, incubation time and temperature (Burt 2004). According to many researchers, it is
found that essential oils are more active against Gram-positive bacteria than Gram-negative bacteria (Shelef 1983;
Holley and Patel 2005). Despite the beneficial antimicrobial action of essential oils, using it in the food matrix remains
a challenge as it leads to undesirable changes in the sensory characteristics of the food. So, the selection of an essential
oil should be able to manage the intricate balance of fulfilling potential antimicrobial activity without negatively af‐
fecting the sensory characteristics.

The objective of this work was to screen the effect of different essential oils against selected LAB and yeast strains as‐
sociated with fermented idli batter, to identify the most efficient to be applied as an antimicrobial agent in the fer‐
mented idli batter system during storage. The evaluation of sensory acceptability and texture of idli prepared from
these treated and stored batter was also carried out.

Materials and methods

Materials

The raw materials, parboiled rice (Oryzae sativa), black gram dal (Phaseolus mungo) and TATA table salt were pro‐
cured from the local market, Mumbai. Essential oils of asafoetida, black pepper (β-caryophylene 10%), clove (eugenol-
85%), coriander (linalool-80%), cumin (cumin aldheydes-40%), fennel (anethole-85%), ginger, garlic (diallyl disul‐
phide-45%) and mustard (allyl isothiocyanate-99%) were purchased from Synthite, Mumbai, India. Sodium chloride,
sodium hydroxide and ethanol were obtained from Merck, Mumbai, India. Plate count agar, deMan, Rogosa and
Sharpe (MRS) agar, and Potato Dextrose agar (PDA) were bought from Hi-Media, Mumbai, India. The microbial cul‐
tures Leuconostoc mesenteroides NCIM 2073, Lactobacillus plantarum NCIM 2084, Lactobacillus acidophilus
NCIM 2903, Lactobacillus casei NCIM 2364 were obtained from NCL, Pune, India. Pediococcus pentosaceus CFR
2123 and Candida versatilis CFR 505 were obtained from CFTRI, Mysore, India.

Determination of the minimum inhibitory concentration by broth dilution method

The broth dilution test was performed to determine MIC using the standard procedure described by Wiegand et al.
(2008). Overnight cultures were suspended in sterile saline and turbidity was adjusted as per McFarland Standard.
Essential oil stock (4000 ppm) solutions were prepared in 90% ethanol. Typically,10–100 µL of diluted stock solution
was taken in sterile test tubes and made up to 1.9 mL by addition of sterile MRS broth for LAB culture and potato dex‐
trose broth (PDB) for yeast. Similarly, control broth was prepared by adding 90% ethanol instead of essential oil stock
solution. A 0.1 mL of inoculum was added to all test tubes to achieve final cell count of 5 × 105 CFU/mL. Test tubes
were incubated at 37 °C for 48 h and turbidity was measured at 625 nm using Spectrophotometer (UV1800, Shimadzu,
Kyoto, Japan). After incubation, 100 µL of broth was plated on to MRS plate for LAB cultures and PDA plate for yeast
cultures. The MRS plates were incubated at 37 °C for 48 h and PDA plate were incubated at 30 °C for 48 h. Based on
the visible growth of LAB and yeast on their respective plates, the biocidal and static effect of essential oils was
determined.

Preparation of batter

Parboiled rice and black gram dal (3:1 (w/w)) were soaked separately in distilled water at 30 °C for 4 h. After draining
off the distilled water, the black gram dal was ground separately to a fine paste whereas rice was ground to a coarse
consistency. Water used for grinding was such that 150 ml of water were used for grinding of 100 g black gram dal and
300 g rice each. These two batters were then mixed, 0.9% table salt was added and the mixed batter was fermented at
30 °C for 12 h (Iyer and Ananthanarayan 2008).

Addition of essential oils in fermented idli batter

To evaluate the sensory acceptance of essential oils of asafetida, black pepper, white pepper, coriander, clove, cumin,
fennel, garlic, ginger, parsley seed, and mustard they were added in 12 h fermented idli batter at different concentra‐
tions based on preliminary trials. Essential oil added idli batter was mixed properly by hand blender (HR1459, Philips)
for 3 min and finally idlis were prepared for sensory analysis. For storage studies, the mustard essential oil was added
at 0.1% (w/w) to the 12 h fermented batter and mixed properly by hand blender for 3 min. The batter was transferred to
clean plastic containers and immediately stored at 30 and 4 °C for further studies.

Evaluation of batter

pH The pH of the idli batter stored at 30 and 4 °C was measured directly using a digital pH meter (Iyer and
Ananthanarayan 2008).

Titratable acidity Typically, 10 g of idli batter was mixed with 20 mL of distilled water and titrated against freshly pre‐
pared 0.1 N NaOH using phenolphthalein as an indicator. The acid content of the idli batter was calculated in terms of
anhydrous lactic acid produced (William 1980).

Viscosity The viscosity (cP) of the control batter and batter with addition of 0.1% (w/w) mustard essential oil was
measured at 20 rpm using LV-4 (64) spindle, Brookfield DV III Rheometer, Middleboro, USA (Iyer and
Ananthanarayan 2008).
Batter volume The batter samples (25 mL) were taken in 100 ml measuring cylinders and stored at 30 °C for 5 days
and 4 °C for 30 days. Batter volume was noted after every 24 h and percentage decrease in batter volume was calcu‐
lated (Nisha et al. 2005).

Whey separation Whey separation was evaluated by measuring separated whey in measuring cylinder containing bat‐
ter sample used for evaluation of batter volume (Nisha et al. 2005).

Microbial Analysis The microbial analysis of batter samples was done to determine the Total Plate Count (TPC), LAB
count and yeast and mold count (YMC), using spread plate method. Sterile plate count agar, MRS agar, PDA was used
for analysis of TPC, LAB, and YMC respectively. 10 g of idli batter was homogenized in 0.9% (w/v) of 100 mL sterile
saline and serial dilutions were prepared. Samples were plated in triplicates, for yeast and mold count the plates were
incubated at 30 °C for 48 h. For TPC and LAB count the plates were incubated at 37 °C for 24 h and the colony form‐
ing units were noted.

Preparation of idli

Typically, 30 g of idli batter (different study variants) were transferred to idli pan and steamed for 10 min. These idlis
were subjected to sensory analysis. Similarly, control batter and mustard essential oil added at 0.1% (w/w) to the batter
were also cooked and evaluated for sensory attributes, bulk density, color, and texture as described below.

Evaluation of idli

Sensory analysis of idli A panel of 15 semi-trained panelists evaluated the control idli and different essential oil incor‐
porated idlis based on a 9-point hedonic scale (ranging from extreme dislike to extreme like) for appearance, texture,
aroma, taste, and overall acceptance.

Bulk density The bulk density (g/cm3) of the idli samples was measured by the seed displacement method using mus‐
tard seed (Nisha et al. 2005).

Color The color was determined by Lab Scan XE system (Hunter Associates Laboratory, Inc., Virginia, USA). The
standard black and white tiles were used to calibrate the instrument. The idli sample was kept inside the cuvette and
both L-value and b value were noted.

Texture analysis The texture of idlis was analyzed by the Texture analyzer (TAX T2i, Stable Microsystems, Surrey,
UK). A cylindrical probe P/36 R was loaded on the 50 kg load cell and it was calibrated by Texture expert software.
The probe was set to 1 mm/s speed and it was allowed to compress the idli sample up to 5 mm depth. The force (N)
needed for compression of idlis was noted.

Statistical analysis

All tests were performed in triplicates and results are expressed as mean ± S.D. Statistical differences between samples
was compared by Duncan’s multiple range test using IBM©SPSS® version 20 at a p value of 0.05.

Results and discussion

Antimicrobial effect of essential oils

The MIC of essential oils and its effect on Leuconostoc mesenteroides, Lactobacillus plantarum, Lactobacillus aci‐
dophilus, Lactobacillus casei, Pediococcus pentosaceus and Candida versatilis are shown in Table 1. Mustard essen‐
tial oil was the only compound which showed a biocidal effect at 40 ppm against Candida versatilis and at 80 ppm
against most LAB strains. Asafoetida, pepper, ginger, and fennel showed a static effect in the range of 200 to 360 ppm.
Lakshmi (2010) reported that pepper essential oil showed the static effect at 250 ppm against staphylococcus aureus,
Bacillus cereus, and Salmonella typhi. In the present study, clove and cumin also showed a static effect by inhibiting
LAB and yeast growth in the range of 400 to 600 ppm. According to Kalemba and Kunicka (2003), clove showed the
static effect at 400 ppm against saccharomyces cerevisiae.

Table 1

Minimum inhibitory concentration (ppm) of various essential oils against LAB and yeast cultures and concentration of essential oils in‐
corporated in batter and accepted in cooked idlis by sensory panelist

Essential oil Effect MIC (ppm)a Upper limit of concentration of essential oils

L.m L.p L.c L.a P.p C.v accepted in idli (ppm)

Asafoetida Static 270 360 270 330 300 270 1000

Black Static 270 330 300 300 300 330 1200

pepper

White Static 300 360 300 330 330 330 1400

pepper

Coriander Static 1500 1500 1125 1125 1000 1000 N.A.

Clove Static 400 500 500 500 500 600 N.A.

Cumin Static 450 550 500 550 450 500 700

Fennel Static 240 200 200 200 200 240 N.A.

Garlic Static 3000 3500 3500 3500 3500 4000 1200

Ginger Static 250 250 250 250 250 225 1000

Parsley Static 1250 1250 1250 1250 1250 1250 N.A.

seeds

Mustard Cidal 80 80 80 60 80 40 1200

L.m, Leuconostoc mesenteroides; L.p, Lactobacillus plantarum; L.c, Lactobacillus casei; L.a, Lactobacillus acidophilus; P.p,
Pediococcus pentosaceus; C.v, Candida versatilis; N.A., Not acceptable at 500 ppm

a
Mean value of triplicate determinations reported with S.D. being negligible

Allyl isothiocyanate (AITC) present in mustard essential oil is well-known for its antimicrobial activity against LAB,
pathogenic microbes and yeast (Shelef 1983; Shofran et al. 1998; Ko et al. 2012). The antibacterial mechanism of
AITC was most similar to polymyxin B, which creates pores on the cell membranes and induces leakage of cellular
metabolites (Lin et al. 2000). According to Ko et al. (2012), the microbial growth of L. plantarum and L. mesen‐
teroides with an addition of 0.1% encapsulated AITC incubated at 30 °C for 24 h was significantly reduced to half of
the control. Shofran et al. (1998) has reported that AITC had a MIC of 50 to 1000 ppm against bacteria and 1–50 ppm
against yeast. The MIC of mustard essential oil determined in this study showed it to be a potential antimicrobial agent
for preservation of idli batter.

Sensory acceptance of essential oils

The sensory acceptance of essential oils in idli (Table 1) shows the upper limit of the concentration of each essential
oil that can be added to the idli batter without affecting the acceptability of idli. Based on the sensory evaluation re‐
sults (supplementary Table 1) fennel, coriander, clove, and parsley seed essential oils were not acceptable at 500 ppm.
Even though fennel and clove essential oils showed good antimicrobial activity, they were not accepted by the sensory
panelists. Black pepper, white pepper, garlic, and mustard essential oils were acceptable up to 1200 ppm, while ginger
essential oil was acceptable up to 1000 ppm. Ginger, asafoetida, white pepper, and black pepper essential oils showed
good sensory acceptance but these essential oils cannot be preferred due to their lower antimicrobial potential. Thus
mustard essential oil was taken forward to determine its antimicrobial efficacy in the preservation of idli batter during
storage.

Efficacy of mustard essential oil in preservation of fermented idli batter

pH and titratable acidity pH and titratable acidity are the major quality parameters which determine the characteristic
sour taste of idli (Shrivastava and Ananthanarayan 2015). The trends of pH and acidity of idli batter incorporated with
0.1% mustard essential oil and control batter stored at 30 °C for 7 days and at 4 °C for 30 days are shown in Fig. 1. The
pH of idli batter with added mustard essential oil was significantly (p < 0.05) higher than that of the control idli batter
at 30 °C for 5 days which clearly indicates that mustard essential oil was more effective in controlling the metabolic
activity of the fermenting microflora in the batter during storage. Likewise, mustard essential oil incorporated batter
stored at 4 °C for 30 days showed significantly higher pH and lower acidity than the control batter stored under similar
conditions, which could be both due to the inhibition of microbial growth by addition of mustard essential oil and
lower temperature of storage. According to Ko et al. (2012), kimchi, a Japanese fermented food incorporated with
AITC (0.2%) stored at 4 and 10 °C for 15 days showed significantly higher pH than the control sample. Furthermore,
in our study, the batter incorporated with mustard essential oil stored at 30 °C after 5 days did not show any significant
difference in pH and acidity from the control batter (Fig. 1a, b). This may be because mustard essential oil is more ef‐
fective at neutral pH and lower temperature. It has been reported that the AITC was more stable at 4 °C than at room
temperature (28–30 °C) and its activity is pH dependent showing effective antimicrobial activity at neutral pH
(Olaimat and Holley 2016).
 Fig. 1

Changes in pH, titratable acidity and viscosity of control idli batter and idli batter with added 0.1% (w/w) mustard essential oil (MEO)
stored at 30 and 4 °C. Data having a common letter are not significantly different (p > 0.05)

Viscosity of idli batter Idli batter contains three phases—solid, liquid and gas. The decrease in viscosity of idli batter
stored at 30 and 4 °C are depicted in Fig. 1e, f. It has been reported that idli batter without the addition of preservative
or stabilizers showed decrease in the viscosity at both ambient (30 °C) and refrigerated storage conditions (Nisha et al.
2005; Chelliah et al. 2016). In the present study, idli batter incorporated with mustard essential oil had a significantly
higher viscosity than the control sample stored at 30 °C for 5 days and at 4 °C for 30 days. The effect of added mustard
essential oil was found to be greater at refrigerated storage condition, especially for the longer time. According to
Reddy et al. (1982), the stability of the structure of idli batter is achieved by entrapment of CO2 by surface active pro‐
teins, holding of leavened gas, while prevention of disruption of foam is majorly achieved by arabinogalactan. It was
also reported that the arabinogalactan showed good stabilization and high viscogenic property around pH 5.0–7.0
(Reddy et al. 1982). The decline in pH of control batter is faster than the batter added with mustard essential oil which
could be one of the reasons for the rapid collapsing of batter system. In the control batter, the rapid decline of viscosity
may be due to liquefaction of starch by amylase enzymes produced by indigenous microbes. However, the addition of
mustard essential oil reduced the metabolic activity of indigenous microbes in the batter which resulted in less acidity
leading to improve the retention of viscosity than control batter.
Batter volume and whey separation Batter volume and whey separation play important role in the texture of the idli.
The decrease in batter volume and increase in whey separation leads to an increase in bulk density and hardness of idli
(Shrivastava and Ananthanarayan 2015). As compared to control batter, the batter with added mustard essential oil
showed significantly (p < 0.05) less decrease in volume under both storage conditions i.e., at 30 and 4 °C (Table 2).
Idli batter with added mustard essential oil showed 15.16 and 12.86% decrease in batter volume when stored at 30 °C
for 5 days and 4 °C for 30 days which was significantly lesser than the control samples. In this study, the addition of
mustard essential oil in idli batter delayed the collapsing of the batter during storage which resulted in lower percent‐
age decrease in batter volume as compared to the control batter.

Table 2

Decrease in batter volume and whey separation in fermented idli batter without (control) and with added mustard essential oil (0.1%)
stored at 30 and 4 °C

Batch Decrease in batter volume (%) Whey separation (%)

Storage Control 0.1% Mustard EO Control 0.1% Mustard EO

30 °C

5 days 21.15 ± 1.04cd 15.16 ± 0.98d 16.10 ± 0.46c 8.00 ± 0.4c

4 °C

5 days 4.25 ± 0.46a 2.66 ± 1.15a 0a 0a

10 days 12.67 ± 1.15b 6.66 ± 0.95b 8.01 ± 1.2b 0a

20 days 19.33 ± 1.05c 10.50 ± 2.10c 15.73 ± 0.42c 4.60 ± 0.41b

30 days 24.16 ± 3.06d 12.86 ± 2.15cd 18.80 ± 0.69d 7.50 ± 0.35c

Mean ± S.D. of triplicate determination of each of three sets of batter. Different letters indicate a significant difference (p < 0.05) within
the same column

The control batter stored at 30 °C for 5 days and 4 °C for 30 days showed 16.10 and 18.80% of whey separation. Batter
containing mustard essential oil stored at 30 °C for 5 days and 4 °C for 30 days showed 8.0 and 7.5% of whey separa‐
tion. No whey separation was observed until 5 days at refrigerated storage for both control and mustard essential oil
added batter (Table 2). Batter incorporated with 0.1% mustard essential oil showed less percentage of whey separation
than control batter throughout the storage at 30 and 4 °C.

Microbial count AITC can inactivate fungal and pathogenic bacteria on cheese, sausages, and chicken breast; it can
even prevent microbial spoilage and act as a strong inhibitor for A. aceti, S. ellipsoideus, S. cerevisiae, and
Mycoderma vini in pickles and sauerkraut (Shelef 1983; Nielsen and Rios 2000; El Fayoumy et al. 2017). The micro‐
bial counts of the batter samples stored at 30 and 4 °C are shown in Table 3. The TPC of control batter increased both
at 30 °C for 5 days and 4 °C for up to 10 days after which slight declination in counts was found on continued storage.
It might be due to the pH tolerance of LAB species at the high concentration of organic acid that resulted in the in‐
crease or maintained LAB count in control batter stored at 30 and 4 °C. From TPC, it is observed that the bacterial
count in the batter containing mustard essential oil stored at 30 and 4 °C was lesser than in the control batter. Idli bat‐
ter with mustard essential oil stored at 30 °C for 5 days showed 2.41 log10 reduction in yeast count compared to the
control batter. Similarly, 1.11 log10 reduction in LAB count compared to the control batter was observed on the third
day, which further increased remarkably. 12 h fermented idli batter showed the pH of 4.95, which may reduce the effi‐
cacy of the mustard essential oil because its antimicrobial activity depends on pH and temperature. It was reported that
inhibition of L. monocytogenes strains by AITC at neutral pH was three times higher than pH 5.0 over 10 d at 4 °C
(Olaimat and Holley 2016). It was reported that addition of AITC (0.1% w/w) in acidified chicken meat shows the de‐
lay in growth of some LAB and aerobic mesophilic bacteria for 2 days (Holley and Patel 2005).

Table 3

Microbial counts (log10 CFU/g) in fermented idli batter without (control) and with added mustard essential oil (0.1%) and stored at 30 and
4 °C

Days Control Mustard essential oil 0.1%

TPC LAB Y&M TPC LAB Y&M

30 °C

12 h 9.20 ± 0.18a 9.11 ± 0.17a 7.94 ± 0.02a 9.13 ± 0.20c 9.01 ± 0.21c 7.77 ± 0.13d

1 9.80 ± 0.06ab 9.50 ± 0.16b 8.28 ± 0.13b 8.97 ± 0.02a 8.52 ± 0.07a 6.54 ± 0.11c

3 9.93 ± 0.02ab 9.76 ± 0.06b 8.66 ± 0.05c 8.84 ± 0.06a 8.65 ± 0.08a 6.25 ± 0.10b

5 10.80 ± 0.60c 10.22 ± 0.24c 8.44 ± 0.12b 9.08 ± 0.05b 8.95 ± 0.07b 6.03 ± 0.09a

4 °C

12 h 8.97 ± 0.21ijk 8.80 ± 0.10i 7.84 ± 0.13jk 8.94 ± 0.32k 8.71 ± 0.21k 7.80 ± 0.18m

5 9.23 ± 0.15k 9.15 ± 0.11j 7.91 ± 0.18k 8.90 ± 0.09k 8.49 ± 0.08j 6.24 ± 0.11l

10 9.18 ± 0.16jk 8.96 ± 0.06ij 7.77 ± 0.14jk 8.87 ± 0.14k 8.43 ± 0.21j 6.00 ± 0.10k

20 8.94 ± 0.06ij 8.72 ± 0.21i 7.60 ± 0.10j 8.60 ± 0.13j 8.09 ± 0.16i 5.71 ± 0.15j

30 8.84 ± 0.12i 8.66 ± 0.26i 7.32 ± 0.10i 8.32 ± 0.11i 7.79 ± 0.10i 5.48 ± 0.09i

Mean ± S.D. of triplicate determination of each of three sets of batter. Different letters indicate a significant difference (p < 0.05) within
the same column

However, essential oil incorporated batter stored at 4 °C for 30 days showed almost 1.84 log10 reduction in yeast count
and 0.87 log10 reduction in LAB count compared to the control batter. Inatsu et al. (2005) reported that the addition of
AITC suppresses the growth of LAB from 2.85 log10 CFU/g to 2.0 log10 CFU/g after 4 days of storage at 10 °C in fer‐
mented Chinese cabbage. Similarly, the coating of the mustard extract on chicken breast reduced the numbers of aero‐
bic bacteria and LAB by 1.1 log10 CFU/g and 1.4 log10 CFU/g, respectively, at 4 °C in 21 days (Olaimat and Holley
2016). In the present study, the addition of mustard essential oil significantly reduced the yeast and LAB count during
storage of idli batter. This will have practical application in augmenting the preservative effect during the storage and
distribution of idli batter where if ideal storage conditions are not maintained then the addition of mustard essential oil
will have an additional inhibitory effect on the metabolic activity of LAB and yeast.

Evaluation of Idli incorporated with mustard essential oil

Idlis made from stored control batter and mustard essential oil incorporated batter (stored at 4 and 30 °C) were ana‐
lyzed for color, texture, and sensory attributes.
Color value of idli The color of the idli was measured in terms of Hunter L (lightness ranging from 0 to 100 indicating
black to white) and b (+ b; yellowness and − b; blueness) values which are shown in Table 4. It was observed that stor‐
age of control batter at 30 °C for 5 days and 4 °C for 30 days resulted in increase in the degree of yellowness and de‐
crease in the degree of whiteness of the idli. The hunter L value and b value of fresh idli were 78.24 and 12.96, respec‐
tively. Idli made from stored batter (at 30 °C for 5 days) with added mustard essential oil showed higher degree of yel‐
lowness and less degree of whiteness than fresh idli. Idli made from the stored batter (at 4 °C for 30 days) with added
mustard essential oil showed L value of 71.89 and b value of 16.57; this indicates a significant decrease in whiteness
and increases in yellowness when compared with the fresh idli.

Table 4

Color analysis, bulk density and hardness of idli prepared from control and batter containing mustard oil (0.1%) stored at 30 and 4 °C

Batch L-value b-value Bulk density (g/cm3) Hardness (N)

Storage Control 0.1% Control 0.1% Control 0.1% Control 0.1%

Mustard EO Mustard EO Mustard EO Mustard EO

12 h fermented 78.24 ± 77.98 ± 0.25d 12.96 ± 13.04 ± 0.15d 0.55 ± 0.57 ± 0.03a 24.33 ± 25.09 ± 2.69d
e a a a
(fresh idli) 0.34 0.19 0.02 2.03

30 °C

5 days 76.63 ± 73.23 ± 0.55b 14.09 ± 15.01 ± 0.19b 0.83 ± 0.76 ± 0.04bc 64.23 ± 56.70 ±
c c c c
0.28 0.17 0.03 3.89 5.93bc

4 °C

5 days 77.74 ± 77.06 ± 0.52c 13.64 ± 14.02 ± 0.06a 0.66 ± 0.59 ± 0.07a 39.83 ± 33.84 ± 3.36a
0.32d 0.32b 0.02b 4.20b

10 days 77.03 ± 76.58 ± 0.71c 13.56 ± 14.93 ± 0.24b 0.84 ± 0.65 ± 0.04a 63.06 ± 36.56 ± 3.13a
0.17c 0.57b 0.05c 4.18c

20 days 75.60 ± 73.18 ± 0.10b 13.83 ± 15.23 ± 0.14b 0.95 ± 0.72 ± 0.02b 75.36 ± 52.11 ± 3.65b
0.25b 0.22bc 0.04d 3.82d

30 days 73.79 ± 71.89 ± 0.15a 14.96 ± 16.57 ± 0.30c 1.12 ± 0.81 ± 0.03c 118.68 ± 62.12 ± 2.08c
0.26a 0.11d 0.02e 5.14e

Value represents the Mean ± S.D. of triplicate determination of each of three sets of batter. Different letters indicate a significant differ‐
ence (p < 0.05) within the same column

Bulk density and hardness of idli Bulk density and hardness are interrelated parameters which determine the texture of
the idli. The hardness of the idli is indicated by the maximum force required to compress the idli. Bulk density and
hardness of idli cooked from the stored batter (at 30 and 4 °C) were found to increase with the time of storage (Table 4
). Bulk densities of control idlis made from batter stored at 4 °C for 10, 20 and 30 days were 0.88, 0.95 and 1.12 g/cm3
respectively. Idli prepared from the batter with added mustard essential oil stored at 4 and 30 °C had significantly less
bulk density and hardness than control idli; this indicates that quality of idli made from the stored batter with added
mustard essential oil was better than the idlis made from stored control batter samples. The minimum force of 24.33 N
was required to compress idli made from optimally (12 h) fermented control batter (fresh idli) and the maximum force
of 118.68 N was shown for idli made from control batter stored at 4 °C for 30 days whereas the force required to com‐
press idli made from mustard essential oil incorporated batter stored at 4 °C for 30 days was only 62.12 N. From this it
may be concluded that mustard essential oil incorporated batter stored at 4 °C gave rise to soft and spongy idlis of ac‐
ceptable texture.

Sensory analysis of idli The results of the sensory analysis indicate that the overall acceptability of idli decreased with
increase in storage of idli batter at 30 and 4 °C (Table 5). In general, increase in the fermentation time of idli batter in‐
creases the degree of sourness in idli. Idli prepared from the batter with added mustard essential oil stored at 4 °C for
10 days significantly scored higher for taste and mouth feel than control idli which shows that the incorporation of
mustard essential oil reduced the sour taste and improved the texture of idli during the storage. According to Ko et al.
(2012), the addition of AITC induced reduction of sour taste and improvement of the texture of Kimchi during fermen‐
tation. The appearance of the idli prepared from the batter with added mustard essential oil stored at 30 °C for 5 days
scored significantly less than the control idli. Control idli made from fermented batter stored at 4 °C for 10 days was
rated as “neither like nor dislike”, due to high sourness and hard texture. The addition of mustard essential oil in the
idli batter stored at 30 °C for 5 days and 4 °C for 30 days showed that the overall acceptability of idlis prepared from
these batters fell in the category of “like moderately” with the score of 6.22 and 6.34 respectively.

Table 5

Sensory analysis of idli prepared from batter containing mustard essential oil (0.1%) stored at 30 and 4 °C

Batch Sensory analysisa

Appearance Aroma Mouth feel Taste After taste Overall acceptability

Control 12 h, fresh 7.86 ± 0.42 7.63 ± 0.81 7.90 ± 1.00 7.63 ± 1.10 7.72 ± 0.97 7.86 ± 0.81

5 days storage

Control, 4 °C 7.59 ± 0.91 7.40 ± 0.43 7.31 ± 0.64 7.50 ± 0.50 7.36 ± 0.71 7.54 ± 0.47

0.1% Mustard EO, 4 °C 7.09 ± 0.46 6.86 ± 0.57 6.83 ± 0.54 6.72 ± 0.87 6.63 ± 0.94 7.13 ± 0.53

0.1% Mustard EO 30 °C 6.40 ± 0.49 6.13 ± 0.71 5.95 ± 0.41 5.95 ± 0.61 6.14 ± 0.85 6.22 ± 0.50

10 days storage

Control, 4 °C 6.42 ± 0.21 6.10 ± 0.41 5.04 ± 0.44 5.12 ± 0.51 5.68 ± 0.29 5.15 ± 0.50

0.1% Mustard EO, 4 °C 6.85 ± 0.23 6.69 ± 0.34 6.69 ± 0.36 6.71 ± 0.42 6.60 ± 0.24 6.70 ± 0.39

20 days storage

Control, 4 °C N.D N.D N.D N.D N.D N.D

0.1% Mustard EO, 4 °C 6.36 ± 0.37 6.56 ± 0.37 6.51 ± 0.46 6.50 ± 0.44 6.49 ± 0.26 6.50 ± 0.29

30 days storage

Control, 4 °C N.D N.D N.D N.D N.D N.D

0.1% Mustard EO, 4 °C 6.08 ± 0.41 6.42 ± 0.31 6.45 ± 0.27 6.27 ± 0.36 6.36 ± 0.45 6.34 ± 0.36

9-point hedonic scale used for sensory analysis: 1—dislike extremely, 2—dislike very much, 3—dislike moderately, 4—dislike slightly, 5
—neither like or dislike, 6—like slightly, 7—like moderately, 8—like very much, 9—like extremely

N.D, not determined because from 10th day itself, it was not acceptable by sensory panelist

a
Mean ± S.D. score of 15 determinations (15 semi trained panelists)
Conclusion

In this present study, it has been demonstrated that mustard essential oil was the potential bio-preservative, among the
other essential oils tested, due to its biocidal effect against selected LAB and yeast strains. The addition of mustard es‐
sential oil at 0.1% (w/w) in idli batter extended the shelf life by reducing LAB and yeast count. The batter with added
mustard essential oil showed significantly higher pH, less titratable acidity, and improved stability. While fermented
idli batter cannot be stored at 30 °C for more than a day, the incorporation of 0.1% mustard essential oil in optimally
(12 h) fermented idli batter extended the shelf life up to 5 days when stored at 30 °C and up to 30 days at 4 °C.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 24 kb)(25K, docx)

Acknowledgements

One of the authors Mr. Baburaj Regubalan is grateful to University Grants commission—Basic Scientific Research
(UGGC-BSR) for the scholarship supported from the Government of India.

Contributor Information

Baburaj Regubalan, Email: baburaj1326@gmail.com.

Laxmi Ananthanarayan, Phone: +91 22 33612506, Email: l.ananthanarayan@ictmumbai.edu.in.

References

1. Burt S. Essential oils: their antibacterial properties and potential applications in foods—a review. Int J Food Microbiol. 2004;94:223–253.
doi: 10.1016/j.ijfoodmicro.2004.03.022. [PubMed] [CrossRef] [Google Scholar]

2. Chelliah R, Ramakrishnan SR, Premkumar D, Antony U. Bio-fortification and shelf-life extension of idli batter using curry leaves (Murraya
koenigii) J Food Sci Technol. 2016;53:2851–2862. doi: 10.1007/s13197-016-2264-2. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

3. Dhamija A (2017) iD Fresh Food has built a brand out of ready-made idli-dosa batter. http://www.forbesindia.com/article/work-in-progress/id-
fresh-food-has-built-a-brand-out-of-readymade-idlidosa-batter/46717/1. Accessed 15 Jan 2018

4. Durgadevi M, Shetty PH. Effect of ingredients on sensory profile of idli. J Food Sci Technol. 2012;51:1773–1783. doi: 10.1007/s13197-012-
0686-z. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

5. El Fayoumy RA, Pendleton P, El-Fallal AA, et al. Allyl isothiocyanate release from edible laminaria japonica for time-dependent growth
deactivation of foodborne pathogens: I: Micrococcus luteus, Bacillus subtilis, and Listeria monocytogenes. Food Bioprocess Technol.
2017;10:1562–1573. doi: 10.1007/s11947-017-1925-0. [CrossRef] [Google Scholar]

6. Holley RA, Patel D. Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiol.
2005;22:273–292. doi: 10.1016/j.fm.2004.08.006. [CrossRef] [Google Scholar]
 7. Inatsu Y, Bari ML, Kawasaki S, Kawamoto S. Effectiveness of some natural antimicrobial compounds in controlling pathogen or spoilage
bacteria in lightly fermented Chinese cabbage. J Food Sci. 2005 [PubMed] [Google Scholar]

8. Iyer BK, Ananthanarayan L. Effect of α-amylase addition on fermentation of idli—a popular south Indian cereal—Legume-based snack food.
LWT Food Sci Technol. 2008;41:1053–1059. doi: 10.1016/j.lwt.2007.07.004. [CrossRef] [Google Scholar]

9. Kalemba D, Kunicka A. Antibacterial and antifungal properties of essential oils. Curr Med Chem. 2003;10:813–829.
doi: 10.2174/0929867033457719. [PubMed] [CrossRef] [Google Scholar]

10. Ko JA, Kim WY, Park HJ. Effects of microencapsulated Allyl isothiocyanate (AITC) on the extension of the shelf-life of Kimchi. Int J Food
Microbiol. 2012;153:92–98. doi: 10.1016/j.ijfoodmicro.2011.10.021. [PubMed] [CrossRef] [Google Scholar]

11. Lakshmi OB. Antibacterial activity of black pepper (Piper nigrum Linn.) with special reference to its mode of action on bacteria. Indian J Nat
Prod Resour. 2010;1(2):213–215. [Google Scholar]

12. Lin CM, Preston JF, III, Wei CI. Antibacterial mechanism of allyl isothiocyanate. J Food Prot. 2000;63:727–734. doi: 10.4315/0362-028X-
63.6.727. [PubMed] [CrossRef] [Google Scholar]

13. Nielsen PV, Rios R. Inhibition of fungal growth on bread by volatile components from spices and herbs, and the possible application in active
packaging, with special emphasis on mustard essential oil. Int J Food Microbiol. 2000;60:219–229. doi: 10.1016/S0168-1605(00)00343-3.
[PubMed] [CrossRef] [Google Scholar]

14. Nisha P, Ananthanarayan L, Singhal RS. Effect of stabilizers on stabilization of idli (traditional south Indian food) batter during storage. Food
Hydrocoll. 2005;19:179–186. doi: 10.1016/j.foodhyd.2004.03.007. [CrossRef] [Google Scholar]

15. Olaimat AN, Holley RA. Inhibition of Listeria monocytogenes on cooked cured chicken breasts by acidified coating containing allyl
isothiocyanate or deodorized oriental mustard extract. Food Microbiol. 2016;57:90–95. doi: 10.1016/j.fm.2016.02.001. [PubMed] [CrossRef]
[Google Scholar]

16. Reddy NR, Sathe K, Pierson MD, Salunkhe DK. Idli, an indian fermented food: a review. J Food Qual. 1982;5:89–101. doi: 10.1111/j.1745-
4557.1982.tb00736.x. [CrossRef] [Google Scholar]

17. Ribeiro-santos R, Andrade M, Sanches-silva A. Essential oils for food application: natural substances with established biological activities. Food
Bioprocess Technol. 2017;10:1562–1573. doi: 10.1007/s11947-017-1925-0. [CrossRef] [Google Scholar]

18. Saravanan C, Shetty PKH. Isolation and characterization of exopolysaccharide from Leuconostoc lactis KC117496 isolated from idli batter. Int J
Biol Macromol. 2016;90:100–106. doi: 10.1016/j.ijbiomac.2015.02.007. [PubMed] [CrossRef] [Google Scholar]

19. Saravanan C, Gopu V, Shetty PH. Diversity and functional characterization of microflora isolated from traditional fermented food idli. J Food Sci
Technol. 2015;52:7425–7432. doi: 10.1007/s13197-015-1791-6. [CrossRef] [Google Scholar]

20. Shelef A. Antimicrobial effects of spices. J Food Saf. 1983;6:29–44. doi: 10.1111/j.1745-4565.1984.tb00477.x. [CrossRef] [Google Scholar]

21. Shofran BG, Purrington ST, Breidt F, Fleming HP. Antimicrobial properties of sinigrin and its hydrolysis products. J Food Sci. 1998;63:621–
624. doi: 10.1111/j.1365-2621.1998.tb15798.x. [CrossRef] [Google Scholar]

22. Shrivastava N, Ananthanarayan L. Use of the backslopping method for accelerated and nutritionally enriched idli fermentation. J Sci Food Agric.
2015;95:2081–2087. doi: 10.1002/jsfa.6923. [PubMed] [CrossRef] [Google Scholar]

23. Sridevi J, Halami PM, Vijayendra SVN. Selection of starter cultures for idli batter fermentation and their effect on quality of idlis. J Food Sci
Technol. 2010;47:557–563. doi: 10.1007/s13197-010-0101-6. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

24. Steinkraus K. Handbook of indigenous fermented foods, revised and expanded. Boca Raton: CRC Press; 1995. [Google Scholar]

25. Wiegand I, Hilpert K, Hancock REW. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial
substances. Nat Protoc. 2008;3:163–175. doi: 10.1038/nprot.2007.521. [PubMed] [CrossRef] [Google Scholar]

26. William H (1980) Official methods of analysis of the Association of Official Analytical Chemists. http://agris.fao.org/agris-search/search.do?
recordID=US19830838740. Accessed 21 Dec 2017