LWT - Food Science and Technology 89 (2018) 155–163

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LWT - Food Science and Technology

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Impact of fermentation and extrusion processing on physicochemical, T

sensory and bioactive properties of rice-black gram mixed flour
Poonam Rani∗, Amit Kumar, Soumya Ranjan Purohit, Pavuluri Srinivasa Rao
Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, West Bengal, 721302, India

A R T I C L E I N F O A B S T R A C T

Keywords: Current study details about the impact of solid state fermentation and subsequent extrusion on the physico-
Bioactive properties chemical, sensory and bioactive properties of the rice-black gram dough. Influence of the solid-state fermen-
Extrusion tation was evaluated, at three levels of yeast concentration (1–3%), sugar (4–8%) and fermentation time (2–6 h).
Fermentation Fermentation resulted in decreased pH and residual sugar, from 6.38 to 5.41 and 1.07 to 0.69%, respectively,
Water absorption index
whereas the titratable acidity was increased from 0.86 to 1.79%, after 6 h of fermentation. Moreover, solid state
Sensory analysis
fermentation for 6 h leads to increased total phenolic content, antioxidant activity and protein content from
29.80 to 44.12 mg GAE/100 g, 3.38–5.10 mg GAE/100 g and 10.36–13.65%, respectively. FTIR analysis showed
decreased crystallinity up on fermentation. Subsequently, dough fermented for 6 h with 3% yeast and 4% sugar
(optimized condition) was considered for the extrusion processing, which leads to increased phenolic content
and antioxidant activity up to 77.3 mg GAE/100 g, and 11.39 mg GAE/100 g, respectively. Expansion ratio, bulk
density, water solubility and water absorption index were determined for product characterization. Furthermore,
extrudate prepared from the fermented flour had slightly higher acceptability (mean score: 4.12) than the un-
fermented counter part (mean score: 3.75) out of 5.

1. Introduction
Application of extrusion technology in the area of product devel-
opment is increasing substantially due to its ease of operation and zero
effluent discharge. Extrusion involves various unit operations like
mixing, shearing, texturizing, size reduction, kneading, shaping,
cooking etc. Moreover, extrusion being the HTST (High temperature
short time) process, exposes food material to high temperature for a
very short time, which limits the detrimental effect of heat on the
various nutritional attributes (Nithya, Bosco, Saravanan,
Mohan, & Alagusundaram, 2016).
Rice is one of the staple cereals being cultivated in more than 100
countries, which serves as major energy source (Ohtsubo, Suzuki,
Yasui, & Kasumi, 2005). The bland taste, white colour and high carbo-
hydrate content make the rice suitable ingredient for the various ready
to eat product development. Furthermore, rice is known to have high
digestibility and hypoallergenicity, which promotes rice starch as best
adoptable material in extrusion technology (Nithya et al., 2016). Gen-
eral practices consider the use of pulses in the diet, in order to overcome
the protein requirement. According to the Indian Pulses and Grain As-
sociation, black gram lentil is a high valued commodity due to its nu-
merous health benefits. It has high protein, fibre and considered as one
of the richest sources of Vitamin B. Thus, combination of the rice and
black gram mix could be considered as nutritious and healthy means for
food product development. Similar recipes already available in case of
South Indian breakfast product such as Idli, in which the bacterial
fermentation of rice and black gram batter, which is carried out to
improve nutritional attributes, texture and flavor (Desikachar et al.,
1960).
It has been documented that fermentation leads to increased free
amino acids, minerals and many other health promoting metabolites.
Additionally, it renders substantial change in taste, flavor and appear-
ance (Hesseltine, Wang, & Regional, 1967). Fermentation also trans-
forms the undesirable components, enhances the nutritive value, and
reduces the energy used for cooking (Blandino, Al-Aseeri, Pandiella,
Cantero, & Webb, 2003; Marshall & Mejia-Lorio, 2012). Apart from this,
fermented foods has been reported to possess increased digestibility and
enhanced protein content, free amino acids, and various extracellular
enzymes (Keishing & Banu, 2015). Moore et al. (2007) found that yeast
treatment resulted in increased protein content by 11–12%.
Altan, McCarthy, and Maskan (2009) studied the effect of extrusion
on the antioxidant activity, total phenolic content of the barley fruit and
vegetable by products, in which barrel temperature (140–160 °C),
screw speed (150–200 rpm) was reported to have antagonistic effect

∗
Corresponding author.
E-mail address: poonam.fpe@gmail.com (P. Rani).

http://dx.doi.org/10.1016/j.lwt.2017.10.050
Received 4 July 2017; Received in revised form 22 October 2017; Accepted 23 October 2017
Available online 25 October 2017
0023-6438/ © 2017 Elsevier Ltd. All rights reserved.
P. Rani et al. LWT - Food Science and Technology 89 (2018) 155–163

and lead to reduced antioxidant activity and phenolic compounds. Table 1

Research related to development of prefermented ready to eat extrudate ANOVA table showing the F values.
has not been addressed so far. Thus, the present study was focused to
Effects Moisture pH TA AOX TPC Protein Glucose
evaluate the effect of the yeast dose, added sugar and fermentation time content
on the physicochemical and bioactive properties of the rice and black
gram dough. Moreover, influence of the extrusion processing on the A-Sugar 7.00* 20.32* 2.51 0.21 1.70 1.73 17.10*
B-Yeast 13.56* 160.99* 67.33* 11.23* 2.04 14.76* 0.0048
physicochemical properties and bioactive compounds was also eval-
C-Time 0.39 487.74* 38.42* 0.37 33.6423* 71.81* 78.84*
uated with the sensory analysis of product. AB 2.68 0.96 2.35 0.059 – 1.81 21.75*
AC 1.12 13.96* 0.16 1.25 – 2.55 21.80*
2. Material and methods BC 1.85 32.02* 6.93* 0.67 – 1.77 0.19
A2 0.80 1.11 2.83 13.01* – .37 15.47*
B2 0.069 0.074 0.074 0.73 – 1.19 2.51
Polished rice flour, black gram and active dry yeast (Saccharomyces C2 19.32* 70.06* 18.09* 1.70 – 28.79* 4.49*
cerevisiae) was procured from the local market, IIT Kharagpur. Polished
rice flour and black gram flour was formed in to dough along with yeast *indicates significant terms for which “p-value” < 0.05; TA: Titratable acidity (% lactic
and sugar for fermentation. Further, the fermentation condition was acid); AOX: Antioxidant activity; TPC: Total Phenolic content.
optimized in order to achieve desirable responses. The optimized pre-
fermented dough was dried and ground in to flour and used as the raw maximization of any response was denoted desirability function (G),
material for the extrusion process. which was obtained by combining the individual desirability values of
different individual di values of variables.
2.1. Fermentation process G = (d1 × d2 × ………..dn)1/ n (3)

The rice flour and black gram flour were mixed in the ratio 3:1 and
dough was prepared, in which 25 g of flour mix was kneaded with
18 ml of yeast and sugar suspensions (Iyer & Ananthanarayan, 2008).
The yeast and sugar suspension was made by mixing different dose of
yeast (1, 2, 3%) and substrate sugar (4, 6, 8%) in 18 mL water. Further,
the dough was incubated at 35 °C and allowed to ferment. To stop the
fermentation at desired time, fermented dough was refrigerated till
further use.
2.2. Experimental design and statistical analysis
A three factor (K), three levels full factorial design was used to
optimize the fermentation process of rice black gram dough for im-
proved nutrition. The design setup consists of 27 (3 × 3 × 3) experi-
mental points. Total numbers of experiments (N) were calculated from
the following equation (1), where k = number of factors and
L = number of levels.
(1)
N= Lk
The three independent variables considered in the current studies
were, dry yeast inoculums (%), sugar content (%) and fermentation
time (h). The relationship between independent variable and responses
were expressed by a quadratic second order polynomial equation and
the generalized form was given as follows in equation (2).
K K k 2.5.1. Moisture content
Y= β0 + ∑ j= 1 β j X j + ∑j=1 βjjXj2 + ∑i ∑ < j=2 βijXiXj (2)
Where, Y is the response; Xi and Xj are variables (i and j range from 1 to
k) and β0 is the model intercept coefficient; βj, βjj, βij are interaction
coefficients of linear, quadratic and the second order terms, respec-
tively; k is the number of independent parameters (k = 3 in this study).
All the experiments were done in triplicates.
Statistical significance of the experimental terms was examined by
analysis of variance (ANOVA) at a confidence level of 95% using the
software Design Expert (version 7.0.0.). This software was also used to
generate the contour plots. The paired t-test was used for finding sig-
nificant difference in sensory properties of the developed products.
2.3. Numerical optimization
The optimization of the various process variables of fermentation
was targeted to maximize the total phenolic content, antioxidant ac-
tivity, protein and to minimize the residual sugar (Table 2). Each re-
sponse was transformed in to dimensionless desirability function (di),
which varies from lower to higher desirability function (0–1). The
Where, di denotes the desirability of the response and n is the number of
responses in the measure. The validity of the developed model was
performed under the optimal conditions as predicted by the model.
2.4. Extrusion processing
The dough developed out of optimized fermentation conditions was
further used for extrusion processing. The fermented dough was dried
in the hot air oven at temperature 45 °C (Ojokoh, Yimin, & Eromosele,
2015) followed by grinding to achieve fine flour form and the moisture
content of the flour was equilibrated to 10% (wb). The flour was fed to
feeder of the co-rotating twin screw extruder (BTPL EB-10 model)
having L/D ratio of 14.4:1. The feeder of the extruder also consist of co-
rotating screw with L/D ratio of 4.5:1. The operating conditions were
barrel temperature 110 °C (die side), and screw speed 300 rpm, circular
die diameter 3 mm producing cylindrical shapes of extrudates (Fig. 4).
Suitable mass flow rate of 11 kg/h was chosen for convenient operation
and kept constant throughout the study. The residence time of the flour
in the extruder for above processing conditions was estimated about
14 s. Extrusion conditions selected for the current study were based on
preliminary experiments.
2.5. Experimental methodology
Moisture content was estimated by convection drying method (BIS,
1978). A known amount of the sample was taken and kept in the oven
at temperature 105 °C till constant weigh achieved.
2.5.2. pH
The pH of the fermented dough was determined by using a digital
pH meter (Make: ADWA; Model: AD8000). Briefly, 3 g of the fermented
dough was diluted with 20 mL of distilled water and mixed thoroughly
using stirrer to make slurry, which was used for pH measurement.
2.5.3. Titratable acidity
Titratable acidity was determined using the standard method, as
described in the literature with some modifications (Sahlin, 1999).
Briefly, 3 g of the dough sample was diluted with 20 ml of the water and
stirred to make a homogenous solution followed by the addition of a
few drops of the phenolphthalein indicator. Further, the solution was
titrated against 0.1 N NaOH till the end point reached. Volume of the
NaOH was recorded and the titratable acidity was expressed as lactic
acid % by using following equation (4).

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Table 2
Constraints table for the optimization.

Name Goal Predicted value Importance Experimental value

(D = 0.82)

Sugar Content (%) Minimize 4 2 4

Yeast Percentage (%) Maximize 3 3 3
Fermentation time (h) Maximize 6 3 6
Moisture Content (% db) Minimize 78.55% 1 80.84
Total Phenolic content Maximize 44.311 4 40.56
(mg GAE/100 g)
Antioxidant activity Maximize 5.10352 4 4.72
(mg GAE/100 g)
Protein content (%) maximize 13.65% 4 12.56
Residual Sugar (%) Minimize 0.50% 2 0.55

Lactic acid % factor 6.25 to obtain protein % on total nitrogen basis.

Volume of NaOH consumed*0.1*Volume made up*equivalent
weight of acid*100
= GODPOD (Glucose Oxidase and Peroxidase) method was used for
Volume of sample taken*weight of sample taken*1000
(4)
2.5.4. Total phenolic content
Ethanolic extract of the sample was obtained by mixing 10 g of the
sample in 20 ml of 80% ethanol. Above solution was stirred to a
homogenous solution and kept for shaking in the shaker for 2 h at 35 °C.
Further, the solution was centrifuged at 10 000 rpm for 15 min and
supernatant was filtered. This extract was then used for total phenolic,
antioxidants estimation. Total phenolic content was determined by the
method described by (Beta, Nam, Dexter, & Sapirstein, 2005) with some
modifications. Briefly, 0.25 ml of sample extract was diluted with
2.75 ml water and 0.25 ml FCR. After 5 min incubation at room tem-
perature 0.75 ml of Na2CO3 (20%) was added. Subsequently, test tube
was kept for 90 min incubation in the dark and absorbance was mea-
sured at 750 nm. Gallic acid standard curve was made to determine the
phenolic content and the results were expressed in the terms of mg
Gallic acid equivalent per 100 g sample (mg GAE/100 g).
2.5.5. Antioxidant activity
Total antioxidants content was determined using the method de-
scribed by (Beta et al., 2005). Briefly, 0.4 ml of the extract was taken
and diluted with 2.65 ml ethanol followed by 0.95 ml DPPH addition.
Further, it was incubated in dark condition for 20 min at room tem-
perature. Subsequently the absorbance was measured using the spec-
trophotometer (Make: HACH; Model: DR2800) at 517 nm. Gallic acid
was used to prepare of standard curve and results were expressed as mg
of GAEAC per 100 g sample by the following equation (5).
GAEAC = (ΔAbssample/ ΔAbsGA)*CGA*(V/W)*100 (5)
GAEAC = Gallic Acid Equivalent Antioxidant Capacity
ΔAbssample = change of the absorbance after addition of sample
ΔAbsGA = change in the absorbance obtained from the standard
calibration curve when same volume of the GA standard solution as
that of sample was added.
CGA = concentration of the GA standard solution (0.01 mg/ml)
V = volume of the centrifuged extract (20 ml)
W = weight of the sample taken (10 g)
2.5.6. Protein content
Protein content was estimated from the organic nitrogen content by
the Kjeldahl method (Said, Harijono, & Kusnadi, 2015). 1 g of dried
sample was taken for the digestion with sulfuric acid, copper sulphate
(II), and potassium sulphate. Subsequently, the digested mixture was
distilled along with 32% NaOH and 4% boric acid followed by titration
against 0.02 N H2SO4. Nitrogen % was multiplied with the protein
2.5.7. Residual sugar
the estimation of the residual sugar present in the sample. A GODPOD
test kit was purchased from Autospan clinical chemistry reagents
(ARKRAY Healthcare pvt. Ltd.) and its prescribed procedure was fol-
lowed for the estimation of residual sugar. The results were expressed in
terms of glucose %.
2.5.8. FTIR analysis
Unfermented and fermented sample (Optimized) samples were
analyzed by the Fourier transform infrared spectroscopy. Finely ground
sample was equilibrated at 14% (db) and formed into pallets with KBr
prior to spectroscopy. The scanning range for the spectrum was 40-
4000 cm−1 (Yang & Tao, 2008).
2.5.9. Water absorption index (WAI) and water solubility index (WSI)
WAI/WSI were determined using the method explained by (Kaur,
Rehal, Singh, Singh, & Kaur, 2014) with slight modifications. Briefly,
2 g of ground extrudate sample was suspended in 20 ml distilled water.
It was stirred for 30 min followed by centrifugation at 8000 rpm for
15 min. Further, the supernatant and sediment was separated and dried.
The WAI and WSI were estimated by the following given equation (6)
and (7).
weight of the wet sediment
WAI(%) =
weight of the dried sediment (6)
weight of solid in supernatant
WSI(%) = × 100
weight of the dry sample (7)
2.5.10. Expansion ratio
Diameter of the extrudate was measured at 5 different random po-
sitions by using Vernier's caliper (Make: Mitutoyo, Model: CD-8″ ASX)
and the expansion ratio was calculated as the ratio of the cross-sectional
diameter of the extrudate to the die diameter as given by equation (8).
Crosssectional diameter of the extrudate
Expansion ratio =
Die opening (8)
2.5.11. Bulk density
Length and diameter of the five randomly selected extrudates pieces
were measured with the digital Vernier's caliper and each of the ex-
trudate was weighed using electronic weighing machine. The bulk
density of the extrudate was expressed in g/cm3, which was calculated
using the following formula (9) and the extrudate was assumed to be
cylindrical shape (Ali, Hanna, & Chinnaswamy, 1996).
4W
Bulk density =
πd 2l (9)

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W = weight of the extrudate (g)
d = diameter of the extrudate (cm)
l = length of the extrudate (cm)
2.6. Sensory evaluation of the product
The descriptive sensory profiling of the puffed products developed
from fermented and unfermented flour at same extrusion conditions
were evaluated based on series of sensory parameters. Both fermented
and unfermented extrudates were incorporated with 2.5% (w/w)
maggie masala. Theses dried extrudates were further sprinkled with
soybean oil (Nupro) and commercial chat masala (5% w/w). The
quantities of spices used were decided by preliminary trails (data not
shown). For the descriptive analysis of sensory attributes, 16 panelists
were selected between the age of 22–30 years. The panelists were se-
lected on the basis of their previous experience in sensory evaluation
and non-smoking behavior was ensured. All the samples were marked
with random three-digit numbers and served simultaneously in dif-
ferent booth. Panelists were asked to score the samples according to 5-
point hedonic scale (1-poor, 2-fair, 3-good, 4-very good, 5-excellent)
(Fadlallah, Tinay, & Babiker, 2010). Panelists were instructed to rinse,
swallow water between the samples. Samples were evaluated for
colour, taste, texture, mouthfeel, aftertaste and overall acceptability.
The average scores of the sensory parameters for both type of ex-
trudates (from fermented and unfermented flour) were calculated and
compared (Sawant, Thakor, Swami, Divate, & Vidyapeeth, 2013). Sta-
tistical significance was assessed using paired t-test. Further, Pearson's
correlation coefficient was determined using XL stat software, in order
to understand the relationship between the overall acceptability and
other sensory attributes.
3. Results and discussion
3.1. Effect of fermentation on physicochemical and bioactive properties of
rice-black gram dough
Effect of various factors and their respective levels has been illu-
strated in Table 1, wherein, Fisher's values (F) have been depicted for
all the factors along with a star superscript to highlight the significant
terms. Higher the “F-value”, greater is the effect of the terms on the
response and lower is the corresponding “p-value”. The “p-value” <
0.05 indicates, the model is significant. Higher F value indicates greater
effect of the factor on respective response.
3.1.1. Moisture content of the fermented dough
Significant influence of sugar % and yeast % on the moisture con-
tent of fermented dough was observed whereas, extended fermentation
time was found to have minimal effect (Table 1). Influence of illustrates
sugar and yeast proportion resulted in reduction of moisture content of
the fermented dough, as presented in Fig. 1 (B). Such reduction in
moisture content was supposed to be due to increased solid addition
prior to fermentation and moisture evaporation from the surface of
dough.
3.1.2. pH and titratable acidity
Effect of the yeast content and fermentation time on the pH and
titratable acidity are shown in Fig. 1C, D and 1(E, F) respectively.
Higher F values for yeast % and fermentation time revealed these two
as most influential factors (p < 0.05) on pH and titratable acidity
(Table 1). Increased proportion of yeast was found to have gradual
reduction in the pH and increment in the titratable acidity. Dough
fermentation at 4% sugar and 3% yeast resulted in lowering of pH from
6.28 (0 h) to 5.41, after 6 h of fermentation. In contrast, the titratable
acidity was found to be increased from 1.01% (lactic acid equivalent) to
1.79%, for the same fermentation time. This reduction in pH and in-
creased acidity is a clear indication about progress of fermentation
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LWT - Food Science and Technology 89 (2018) 155–163
within the dough. The increment in titratable acidity supposed to be
result of conversion of carbohydrate to simpler sugar and subsequent
transformation into mixture organic acids like citric acid, lactic acid,
acetic acid, gluconic acid etc., ethanol, carbon dioxide. Similar result
has been reported by many researchers, in which the increased level of
organic with time was reported in Idli batter (Kaw & Mabesa, 1987).
3.1.3. Residual sugar
The fermentation time and added glucose content affects the re-
sidual sugar content significantly (p < 0.05) as observed from the
ANOVA Table 1. Fermentable sugar was found to be decrease with
fermentation time and yeast content due to the consumption of car-
bohydrate fraction and subsequent conversion into alcohol and organic
acids. There has been a decrease in fermentable sugar from 1.14 to
0.50% after 6 h of fermentation, as recorded with 3% yeast. Reduced
titratable acidity was also found supportive for above variation
(Fig. 1F), which was inverse to the reduced residual sugar content
(Fig. 1H). The pattern of residual sugar decreases in the fermenting
dough at different fermentation time was found similar to results re-
ported by Kaw and Mabesa (1987).
3.1.4. Total phenolic content
Influence of yeast%, sugar% and fermentation time on total phe-
nolic content of fermented dough has been depicted in Fig. 2A. In-
creased fermentation time (p < 0.05) caused gradual increase in the
total phenolic content from 32.22 of control (unfermented dough) to
36.18, 40.15, 44.12 mg GAE/100 g sample after fermentation time of 2,
4, 6 h, respectively while the sugar and yeast level was set at 4 and 3%.
Such enhancement in the total phenolic content is expected to be
caused due to metabolic and enzymatic action of yeast on the bound
phenolic compounds linked to the insoluble part of the cell wall, which
remain cross-linked with polysaccharides and lignin. It is expected that,
the enzymes produced by the microorganism, breaks down the che-
mical compounds of the plants cell matrices that results in the hydro-
lysis of the ester bonds and causes oxidation of lignin (Huynh, Van
Camp, Smagghe, & Raes, 2014). Such oxidation leads to release of free
and bound phenolic compounds. Increase of 50–100% in the total
phenolic content has been observed by the Moore et al. (2007) during
fermentation of hard wheat bran.
3.1.5. Antioxidants activity
Effect of yeast %, sugar% and fermentation time was evaluated on
the antioxidants activity of fermented dough (Fig. 2B and C). Increased
specific inhibition for the DPPH and antioxidant activity can also be
used interchangeably to denote the scavenging power for the free ra-
dicals. Antioxidants activity was found to be increase from 4.10 to
5.10 mg GAE/100 g sample after 6 h fermentation, with varying initial
yeast content of 1%–3%. Increase in fermentation time and yeast con-
tent was found to have an additive effect on antioxidant activity,
however, influence of sugar was observed as minimal. Such increased
antioxidants activity was expected to be caused by release of bioactive
compounds from the dough and extracellular compounds released by
yeast cells. Additionally, increased phenolic content reported in the
current study also supports the reasoning behind enhanced antioxidant
activity of the fermented dough. Similar to total phenolic content,
Moore et al. (2007) also reported that fermentation of wheat bran
caused significant increase in DPPH scavenging capacity by 50–100%.
3.1.6. Protein content
The total protein content of the fermented dough was found to be
increased with fermentation time (p < 0.05) (Fig. 2D and E). Max-
imum protein content of 13.65% was recorded in case of the sample
fermented for 6 h with 3% yeast content. As per literature yeast cell
contain 10% protein on the dry basis, hence yeast proliferation also
expected to cause increase in the total protein content (Kannan,
Chelliah, Rajamanickam, Venkatraman, & Antony, 2015). About 12%
P. Rani et al. LWT - Food Science and Technology 89 (2018) 155–163

Fig. 1. Effect of fermentation conditions on (A) Moisture content of

unfermented dough (B) Moisture content of 6 h-fermented dough
(C) pH of the unfermented flour (D) pH of 6 h-fermented flour (E)
Titratable acidity of unfermented dough (F) Titratable acidity of
6 h-fermented dough (G) Residual Sugar at4% added sugar (H)
Residual Sugar at8% added sugar.

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Fig. 2. Effect of fermentation conditions on

(A) Total phenolic content (B) Antioxidant
activity at 4% sugar (C) Antioxidant ac-
tivity at 8% sugar (D) Protein content at 4%
sugar (E) Protein content at 8% sugar.

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P. Rani et al.
Fig. 3. FTIR spectrum (a) Unfermented flour ( ) and (b) fermented dough (————).
increase in the protein content of the fermenting batter of wheat bran
was also reported by Moore et al. (2007).
3.1.7. FTIR analysis
FTIR analysis was carried out to identify the change and modifica-
tion in the functional groups and starch crystallinity after the process of
fermentation. FTIR spectrum of fermented and unfermented samples
were recorded using FTIR spectrometer, along the wavenumber ranging
from 400 to 4000 cm−1 (Fig. 3). The result revealed that there was no
significant difference in the peaks position, which signifies limited ef-
fect of fermenetation on functional groups. However, slight variation in
intensity ratio of 1047:1022 was noticed as 0.94 and 0.925 for un-
fermented and fermented dough, respctively. In the FTIR spectrum,
absorbance at wavenumber 1047 cm−1 indicates the crystalline or or-
dered structure of starch and absorbance at 1022 cm−1 indicates the
amorphous fraction (Yang & Tao, 2008). Reduced intensity ratio of
1047:1022 for fermented dough (0.925) against unfermented sample
(0.94) indicates about the reduced crystalline fraction possibly due to
fermentation. Lactic acid fermentation of rice flour for 24 h at 30 °C
caused gradual disappearance of crystalline structure of starch
(Yang & Tao, 2008). Another study of fermentation of rice flour for 48 h
indicated minimal variation in the position of peaks, however, they
reported occourance of rice starch modification. (Said et al., 2015).
3.2. Optimization and validation
The Derringer's prediction tool was employed to determine the
optimum process condition to obtain desirable responses under various
constraints, as mentioned in Table 2. Higher amount of bioactive
compounds, protein with minimum amount of the glucose content were
targeted while optimizing the fermentation process. Since, longer fer-
mentation time results in increased bioactive compounds level, the
fermentation time was set as maximum. The optimum processing con-
ditions were found to be 3% yeast, fermentation time of 6 h and 4%
sugar, and responses shown in Table 2 with a desirability value of 0.82.
Further, the prediction was validated by conducting real time experi-
ment in triplicates and the results were found close to the predicted
values, suggesting that full factorial design could be effectively used to
optimize the present fermentation condition to enhance the release of
bioactive properties.
3.3. Effect of extrusion on physicochemical and bioactive properties of the
prefermented extrudate
3.3.1. Expansion ratio(ER)
Expansion is one of the important characteristics for the extruded
puffed product. It affects the quality parameters of the extrudate like
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LWT - Food Science and Technology 89 (2018) 155–163
Table 3
Physical and bioactive properties of the extrudates.
Parameter Extrudate
Expansion ratio 3.96 ± 0.088
Bulk density (g/cm3) 0.0909 ± 0.0036
Water absorption index (g/g) 8.53 ± 0.0256
Water solubility index (%) 21.17 ± 0.373
Total phenolic content (mg GAE/100 g) 77.38 ± 1.98
Antioxidant activity (mg GAE/100 g) 11.39 ± 0.18
crispiness and water absorption behavior. Extrusion of the fermented
flour caused about 4 times expansion of the extrudate at given extrusion
operating conditions (Table 3). This could be due to large difference in
the pressure inside and outside of the extruder, which led to the for-
mation of the air pockets and resulted into porous puffed texture. The
expansion ratio obtained for extrudate product is in the range, which is
known to have ideal degree of puffing for texture acceptability (C.
Charunuch, Limsangouan, Prasert, & Wongkrajang, 2014).
3.3.2. Bulk density
Bulk density shows an inverse relationship with expansion ratio.
Apparent density of the extrudates depends upon how efficiently, mo-
lecules are packed together. Generally, low density expanded extruded
products are considered as desirable to obtain crispy and highly ex-
panded products. Bulk density of the prefermented extrudate was found
out to be 0.0909 g/cm3, which well corresponds with its expansion
ratio and indicative of its lower weight per unit volume. The values of
the expansion and bulk density calculated in the present study fits into
the desirable range (longitudinal expansion indices > 2.5, and unit
bulk density < 0.2 g/cm3) to obtain crispy snacks (da Silva, Ascheri,
Carvalho, Takeiti, & Berrios, 2014). The feed moisture content for the
extrusion processing was 10% dry basis, which is expected to be the
main factor for such lighter product. However, other parameters like,
barrel temperature, raw material composition are also consider to be
governing factors (Nithya et al., 2016).
3.3.3. WAI and WSI
WAI is an indicator of the degree of starch gelatinization, which
enhances the ability of the extrudate to absorb water. Hydration
properties of the extruded products are among the functional properties
that governs the quality of the developed product. Product with too
high WAI will have a greater ability to absorb water and tendency to
become soggy easily, which is undesirable. Similarly, too high WSI of
the product indicates stickiness in the product. Hence these properties
should be defined for the extruded product. The gel forming capacity of
the fermented rice black gram mix flour was found to be
8.53 ± 0.02 g/g, which was remarkably higher than the extrudate
products without any fermentation. The value of the WAI obtained in
the current study corroborate with the findings of wheat flour extrudate
for the screw speed in the range of 200–300 rpm (Schmid, Dolan, & Ng,
2005). Molecular components of starch are degraded which results in
increased soluble components. The WSI of the fermented extrudate was
found 21.175 ± 0.373%. The value of the WSI was found similar to the
range of WSI of brown rice reported by (Charunuch,
Boonyasirikool, & Tiengpook, 2003).
3.3.4. Total phenolic content
Extrusion of the prefermented flour led to a sharp increase in the
total phenolic content of the extrudate. Interestingly, extrusion pro-
cessing was found to enhance the total phenol content of the extrudate
as 77.38 mg GAE/100 g. This significant increase in the phenol content
is probably due to liberation of bound phenolic compounds due to
shearing effect of extrusion. Ozer, Herken, Güzel, Ainsworth and
Ibanoǧlu (2006) studied the effect of the extrusion parameters on the
extruded snacks and they reported minimal change in the phenolic
P. Rani et al.
content. However, findings of the current study illustrate that extrusion
processing enhances the total phenol, which could be due to extended
extraction effect of co-rotating twin screw under high temperature and
pressure.
3.3.5. Antioxidant activity
Increased phenolic content extraction must have increased the
specific inhibition for the DPPH, hence increasing the antioxidant ac-
tivity. Similar to phenolic content, antioxidant activity was also found
to be increased to 11.39 mg GAE/100 g after extrusion. This could be
due to depolymerization effect of extrusion on long polyphenolic chain,
which renders enhanced activity. However, in a contrasting study, Ruiz-
Gutiérrez et al. (2015) highlighted a decrease in the bioactive com-
pounds after extrusion process in the red cactus pear powder at 140 °C
barrel temperature. This difference could be aroused due to variation in
the residence time and temperature of the barrel.
3.3.6. Sensory evaluation of the puffed extrudates
The puffed extrudates produced at optimized processing conditions
from the fermented flour and unfermented flour were evaluated by a
semi trained panelist. The averages score of the all the attributes and
overall acceptability has been represented in the form of a graph
(Fig. 4) Although both the products were in the acceptable range
(Good), but the fermented flour extrudate had superior acceptability
(mean score: 4.125 ± 0.24) than the extrudate prepared from un-
fermented flour (mean score: 3.750 ± 0.19). In addition to this, based
on the average scores of the panelist, fermented extrudate had better
162
LWT - Food Science and Technology 89 (2018) 155–163
Fig. 4. Representation of the sensory profile of the puffed
extrudate; (a) Fermented flour extrudates ( ); (b)
Unfermented flour extrudates ( ).
Fig. 5. Pictorial illustrations of developed puffed extrudates
(A) Fermented flour extrudates (B) Unfermented flour ex-
trudates.
taste (mean score: 4.125), texture (4.187), mouthfeel (4.25) and
aftertaste (4.125). The mean score of the unfermented flour extrudate
were taste (3.75), texture (3.75), mouthfeel (3.9375), and aftertaste
(3.75). However, statistical analysis showed no significant difference
(t = 2.12; p > 0.05) in the overall acceptability of these two products
as analyzed by the paired t-test. Individual sensory attributes were also
found have insignificant difference, except texture. Therefore, it is
clearly evidential about the impact of fermentation on bringing super-
iority in texture. Furthermore, the overall acceptability of unfermented
extrudates was found to be mainly related to texture (r = 0.75), fol-
lowed by followed by taste (r = 0.71) and mouth feel (r = 0.60).
However, in case of fermented extrudates, mouth feel, texture, and after
taste were found equally correlated (r = 0.69–0.64) and collectively
forms a cluster of factor, which was responsible for the overall ac-
ceptability. Being the snack product, it was oblivious that texture has to
play important role in sensory preference and it was true in case of
unfermented sample. However, correlation analysis of fermented
sample showed greater importance of mouth feel (r = 0.69), which can
be define by combined experience of physical and chemical attributes
of the extrudates. Thus, it can be understood that, there was develop-
ment of some flavor compounds, which enhances the mouth feel in case
of fermented extrudates and resulted in higher overall acceptability of
the product. In both the cases, colour was adjudged to have least im-
portance, as far as over all acceptability of the product is concerned. A
pictorial illustration of the developed product has been shown in Fig. 5.
P. Rani et al.
4. Conclusions
Above study made clear that, rice and black gram mix could be an
ideal mixture for new product development based on their suitability
for processing nutritional attributes. Moreover, fermentation for 6 h
resulted in enhanced nutritional and functional attributes. Subsequent
drying and extrusion leads to further improvement in total phenolic
content, antioxidant activity along with other physicochemical prop-
erties. Moreover, according to the sensory test analysis, extrudates from
fermented flour had slightly higher overall acceptability over the ex-
trudates from unfermented flour. However, statistically sensory attri-
butes of both the products are not significantly different. Thus, pre-
fermentation and extrusion could be a better option to improve the
nutritional and sensory acceptability in cereal based products and for
development of new products.
Acknowledgement
First author is thankful to MHRD, Govt of India, for providing
scholarship during M. Tech degree. Assistance provided by Mr. Nirmal
Dolai during experiments and extruder operation has been acknowl-
edged.
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