<Microbial changes during porridge fermentation of maize and some South

Indian millets> Comment [V1]: A4 page 1” margin all around

Single line space, one line gap after title,

Geetha, T. Gnanam, G. and Maheswaran, M. Comment [V2]: Title (Times New Roaman 14
point Bold & Centred
School of Biosciences and technology, VIT University, Vellore - 632014, TamilNadu, India
geetha@gmail.com; +91-22-12345678 Comment [V3]: Authors Times New Roman 11;
Centered,

Comment [V4]: Affliation

Comment [V5]: Email and telephone

Abstract
Comment [V6]: Subtitle after two line gap;
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Porridge (Koozhu) is a popular South Indian traditional food made from locally available grains. The
study was undertaken to investigate microbial succession and biochemical changes occurring during
natural fermentation of four different millet grains viz., Finger millet (Eleucine corocana), Pearl millet
(Pennisitum typhoides), Sorghum (Sorghum bicolor) and Maize (Zea mays). Primary fermentation is
allowed for 20 h and samples were analysed at five hour interval for microbial and biochemical changes.

In all grains, Lactic acid bacteria (LAB) and total bacteria counts were found to increase throughout
fermentation time with significant increase in titrable acidity and concomitant decrease in pH till 20 h.
Starch hydrolysers were the primary players in the initial hours (0 -10 h) of fermentation in all grains,
while LAB dominated the fermentation during later hours (10 -15 h). Except in pearl millet Enterobacter
population was increasing in initial hours but after 15 h of fermentation saturation attained. The
population of yeasts was found to be constant throughout the fermentation period. Significant increase in
reducing sugars was observed at 15 h of fermentation without any change in total sugar level. Protein
content was found to increase in Maize, while in other grains there is no significant variation. This study
identified dominant microbes which can be used for developing functional starters to enhance the product
quality. Comment [V7]: Body Text, Times New Roman,
11, left-aligned, no hyphenation, 1.5 line space

Keywords: Fermentation. Lactic acid bacteria. Yeast. Enterobacter. Starch hydrolysers.

Introduction Comment [V8]: Subtitle after two line gap;

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Fermentation of cereals for various culinary preparations is an age old practice. Various fermented cereal
based preparations are available and they contribute to about one-third of the diet worldwide (Campbell
and Platt 1994). In African and Asian countries, fermentation is used extensively for cereals food
preparations. Fermentation has positive influence on cereals (Simango 1997, Gobbetti et al. 1994,
Olasupo et al. 1996), which is well recognized and accepted from ancient times. The health benefits, taste
and odour of the fermented foods depend on the microbes involved in the fermentation which in turn
depend on substrate and fermentation conditions. Traditional fermented foods have received extensive
scientific attention and many traditional cereal preparations have been analyzed for its microbiological,

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enzymylogical and biochemical changes (Odunfa et al. 2001, Omemu et al. 2007, Mutasa and Ayebo
1993). Almost all traditional cereal preparations start for natural fermentation which comprises of mixed
microbial cultures. Among various fermented food preparation, porridges occupy important position in
African and Asian countries. In South India porridge is made from local varieties of cumbu (Pennisitum
typhoides), ragi (Eleucine corocana) and maize (Zea mays) and was popularly called as koozhu/kanji.
Koozhu has long history in South India and associated with culture and tradition. It is well known for its
flavor and nutritional quality but largely caters to village and physical labour sections in semi-urban and
urban areas. It is also becoming popular among other sections requiring alternative carbohydrate sources
for rice and wheat.

Materials and methods

Chemicals: All the chemicals and reagents used in the study were from Hi Media chemicals, Mumbai and
Sigma chemicals, Bangalore.

Sample preparation: Commonly used grains viz., Finger millet, Pearl millet, Sorghum and Maize were
used in this study. Grains were purchased from Vellore market (Tamilnadu), freed from foreign particles;
milled into flour and stored at room temperature. Natural fermentation was carried out by mixing 25 g of
the milled flour in water in the ratio of 1:2. Drinking water was used in fermentation. The mixture was
incubated at room temperature.

Sampling: Samples were collected every 5 h interval till 20 h in sterile glass vials and stored at 4 oC for
further analysis.

Enumeration of microorganisms: At each sampling time, 1 g of sample was weighed into 0.84% sterile
saline. Appropriate serial dilutions were made and plated on the following media for microbial

Results and Discussion

Analysis of microorganisms involved in fermentation at different hours: Natural fermentation of

grains used in the study exhibited different microbial groups but no significant variation of microbial
groups was observed. Figure 1, 2, 3 and 4 represents microbial succession during natural fermentation of
finger millet, pearl millet, maize and sorghum respectively. In all the grains, there was a gradual increase
in the population of lactic acid bacteria (LAB) till 20 h . Average population on MRS agar plates varied
from 0-10, 0.5-9.0, 3.1-8.9, and 2.6-8.5 log CFU/g of fermented finger millet, pearl millet, maize and
sorghum respectively. Previous reports on cereal traditional preparation stated the involvement LAB in

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cereal based preparations (Omemu et al. 2007, Odunfa et al. 2001, Kasseem, 1999) and results of this
study are in accordance. All the grains used in this study acted as good substrates for Lactic acid bateria.

Two way Anova test (P<0.05) indicated significant increase in LAB population from 10 h of fermentation
which may be due to the fact that starch content in the grains hindered LAB group to act on the substrate
during the initial hours. Hence starch hydrolysers play major role at the initial hours of fermentation (0-10
h) of grains. Their count at the initial hours was high when compared with other groups, but there is no
significant increase in counts during fermentation, also there is any marked variation in count among
grains. There were limited reports on the role of starch hydrolysers in fermentation of grains; however
some of the findings stated the role of amylolytic lactic acid bacteria during fermentation (Olasupo et al.
1996). The result of this study bring out the importance of starch hydrolysers; though the count found to
be low, their contribution found to be significant in the initial hours of fermentation, helps in softening of
grains flour; hydrolyse starch to reducing sugars making way for Lactic acid bacteria. Correlation analysis
showed that in finger millet and pearl millet less correlation (0.4 and 0.3) exists between starch hydrolyser
and Lactic acid bacteria while in maize and sorghum negative correlation prevails (Table 1). As Lactic
acid bacteria takes over the fermentation process converting sugar to acid, pH of the medium becomes
acidic which is unsuitable for starch hydrolysers.

Table 1. Titrable acidity at different fermentation hours

Fermentation

Hour (h) Finger millet Pearl millet Maize Sorghum

0 0.56 ± 0.15 a 1.36 ± 0.2 a 1.06 ± 0.2 a 1.23 ± 0.2 a

5 0.8 ± 0.52 a 2.16 ± 0.05 a 1.23 ± 0.15 a 1.06 ± 0.05 a

10 1.3 ± 0.1 a 2.76 ± 0.2 a 1.5 ± 0.1 a 2.1 ± 0.1 a

15 1.8 ± 0.11 b 4.2 ± 0.4 b 2.06 ± 0.11 b 2.96 ± 0.05 b

20 2.53 ± 0.1 c 5.9 ± 1.4 c 2.5 ± 0.1 c 3.83 ± 0.15 c

• Values are means of triplicate samples (±SD). Means not sharing a common letter in a column are
significantly different at P < 0.05 as assessed by Duncan’s multiple range test.

In addition to sugar changes effect of fermentation on changes in protein content of millets was
also looked in. There was no significant variation in protein concentration, till 10 h however during 15 h
significant increase in protein concentration was observed which attained saturation at 20 h. This finding
may be explained by the fact that increases in Lactic acid bacteria group effects the protein increase at 15

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h (Figure 1). Previous work on pearl millet fermentation conducted by Maha AM Ali (2003) also
reported variation in protein content due to fermentation during the initial hours. In Maize, protein
changes were statistically significant compared to other grains. While in others no significant difference
recorded. Similar reports were given by Nabila et al. (2000) in Maize fermentation.

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10 Total bacteria

8 Total LAB

Total
log CFU/g 6 Enterobacter
Starch
4 hydrolysers

0
0 5 10 15 20 25
Fermentation hour (h)

Figure 1 Microbial succession at different fermentation hours of finger millet Comment [V9]: Graph title in the bottom; bold
left aligned; chart no border with legends + X/Y axes
titles and clear black lines with patterned / colored
Conclusion symbols

The present study has shown that primary fermentation of common used grains for porridge preparation
includes various groups of microorganisms predominantly starch degrading organisms and lactic acid
bacteria. Also in this study we have reported some of the biochemical activities of the organisms
involved. In the initial hours, starch form the major source for microbes which is evident in increase in
reducing sugar content, in later part conversion of sugars results in acid production. Though microbial
profile found to be similar in all the grains, sugar transformations, and protein changes depends on the
composition of grains. The findings will help to improve the fermentation processes especially isolation
and developing a consortium for natural fermentation, value addition by manipulating fermentation flora
are being looked.

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