LWT - Food Science and Technology 122 (2020) 109002

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LWT - Food Science and Technology

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Characterization of microbial polysaccharides and prebiotic enrichment of T

wheat bread with pullulan
S. NithyaBalaSundaria, V. Niveditab, M. Chakravarthyb, G. Srisowmeyab, Usha Antonyc,
G. Nandhini Devb,∗
a
Sri Shakthi Institute of Engineering and Technology, Coimbatore, 641062, India
b
Centre for Food Technology, Anna University, Chennai, 600025, India
c
College of Fish Nutrition and Food Technology, TNJFU, Madhavaram, Chennai, 600051, India

A R T I C LE I N FO A B S T R A C T

Keywords: The prebiotic efficacy of microbial polysaccharides pullulan, xanthan, and gellan was examined by in vitro
Xanthan digestion and in vitro fermentation. In vitro digestion study showed that pullulan exhibited a lower degree of
Pullulan hydrolysis (DOH) than xanthan and gellan. In vitro fermentation study showed that pullulan has improved the
Gellan stimulating effect on the growth of probiotics. Thermo-gravimetric report showed the possible application of
Prebiotics
pullulan in bakery products. In this study, pullulan enriched bread formulations at varying concentrations with
Bread
both refined wheat and wheat-based bread was done. Pullulan showed a positive correlation with probiotic
growth and a negative correlation with water absorption. The nutritive value and physicochemical properties of
the bread varieties were investigated. Bread samples with 2% pullulan exhibited most acceptable sensorial at-
tributes, whereas the formulations with 8% and 10% pullulan were found to be much softer and darker. The in-
vitro digestibility and fermentation of bread samples showed that Pullulan enriched breads were more resistant
to hydrolysis by AGHJ than the standard bread samples. Prebiotic efficacy measured using Prebiotic Activity
Score (PAS) was higher for pullulan among other polysaccharides and pullulan enriched breads against standard
bread.

1. Introduction for formulating foods of choice. Though hydrocolloids are frequently

The increasing concern for health and wellbeing has pioneered the
significance of functional foods in recent times. Functional foods exhibit
a positive effect on health beyond providing nutrition. Prebiotics and
probiotics are major functional food ingredients that aid in improving
and restoring health. Among the probiotics, Bifidobacterium and
Lactobacillus are widely accepted and found capacity in food industries
for various purposes. The growth and occurrence of probiotics are in-
fluenced by many factors such as antibiotics, illness, stress, ageing and
lifestyle (Gerritsen, Smidt, Rijkers, & De Vos, 2011; Woodmansey,
2007). A widely accepted potential approach for improving the gut
microfloral population is by consuming specialized food ingredients
known as prebiotics (Rycroft, Jones, Gibson, & Rastall, 2001). Pre-
biotics are non-digestible food ingredient that benefits the host by se-
lectively stimulating the growth and activity of beneficial colon bac-
teria.
Hydrocolloids, diverse group of long-chain polysaccharides possess
versatile functional properties that make it popular in the food industry
used in the food industry, most of the hydrocolloids do not present any
nutraceutical properties. The growing demand for functional food in-
gredients and the persistent studies that authorize the different health
benefits of prebiotics makes it significant to discover potential and
cheap sources of compounds that gratify the virtuous properties of an
efficient prebiotic. Inulin, a popular commercial prebiotic of hydro-
colloid origin holds the root for the idea of investigating the prebiotic
efficacy of the polysaccharides.
This study was an attempt to investigate the prebiotic efficacy (PE)
of three microbial polysaccharides (xanthan, pullulan and gellan) and
to formulate a prebiotic microbial polysaccharide enriched bread pro-
duct. Xanthan is an extracellular polysaccharide produced by the bac-
terium Xanthomonas campestris (Causse et al., 2013). Pullulan is extra-
cellular homopolysaccharide of glucose produced by many species of
the fungus Aureobasidium, specifically A. pullulans (Kennedy & Panesar,
2005). Pullulan easily dissolves in cold or hot water to form a 94 stable,
viscous solution that does not gel and stable over a wide range of pH
and heat Gellan gum is a polysaccharide produced by the

∗
Corresponding author.
E-mail address: projectsagnlabs@gmail.com (G. Nandhini Dev).

https://doi.org/10.1016/j.lwt.2019.109002
Received 24 August 2019; Received in revised form 26 December 2019; Accepted 28 December 2019
Available online 02 January 2020
0023-6438/ © 2020 Elsevier Ltd. All rights reserved.
S. NithyaBalaSundari, et al.
microorganism Sphingomonas elodea (Kuo, Mort, & Dell, 1986). The
ability of the polysaccharides to satisfy the qualities of a prebiotic was
assessed using in vitro digestibility and in vitro fermentation. The
prebiotic activity score (PAS) was evaluated for the polysaccharides
with inulin as a reference. The polysaccharides that satisfied the ideal
functions of a prebiotic and performed comparable to the commercial
standard (inulin) was further incorporated in a bakery product (bread)
with the intention of amplifying the acceptability and availability of the
functional food as breads are one of the most consumed products
among baked foods (Bakke & Vickers, 2007). The characterization and
the degree of acceptance of the prebiotic enriched breads (PEB) were
evaluated. This study holds significance in the detail of formulating PEB
varieties, especially pullulan enriched breads that have been reported
the least despite its excellent prebiotic activity.
2. Materials & methods
2.1. Materials
Pullulan and gellan were procured from Kumar organic products
Ltd, Bangalore, India. Xanthan and Inulin were procured from
Bakersville, India Pvt. Ltd, Indore, India. All reagents used for analysis
were of analytical grade and were purchased from Himedia laboratories
private limited, Mumbai, India. The raw materials for the bread were
purchased from the local supermarket, Chennai, India. The strains
Lactobacillus acidophilus (ATCC 4356) and Escherichia coli (ATCC 8739)
were purchased from ATCC.
2.2. Characterization of microbial polysaccharides
The competence of the microbial polysaccharides (A - xanthan, B -
pullulan and C - gellan) to endure the gastric acid condition was ex-
amined in vitro by adopting the method described by (X. Wang et al.,
2015). The pH of the Artificial Human gastric juice (AHGJ)
(Supplementary Table 1) was varied to pH 2, 3, and 4 using 1M HCl.
2.5 mL of 10% sample A, B, C and D (Inulin) was subjected to AHGJ
maintained at pH 2, 3 and 4 and incubated at 37 °C for 6 h. The re-
sistance of the polysaccharides A, B, C and D to the gastric acid con-
dition was evaluated by the degree of hydrolysis (DOH). Aliquots of the
samples were taken every 2h for 6h to determine the concentration of
the initial and the final reducing sugar (DNS method) (Robertson et al.,
2001)and total sugar (Phenol-sulfuric acid method)(Dubois, Gilles,
Hamilton, Rebers, & Smith, 1956). The DOH was calculated as
reducing sugar released  of the ash was calculated as crude fibre(Kalnina, Rakcejeva, Gramatina,
DOH (%) = × 100
total sugar − initial reducing sugar
Where
Reducing sugar released = [(concentration of reducing sugar at time t)
– (initial concentration of reducing sugar)]
The PE of the polysaccharides (A, B, C and D) were examined in
vitro using the method reported by (Shalini, Abinaya, Saranya, &
Antony, 2017). Distinct solutions of polysaccharides (10% w/v in
water) - xanthan (A), pullulan (B), gellan (C), inulin (D) and dextrose
were prepared. To the sterilized carbohydrate-free MRS basal media
(CFMBM) (Supplementary Table 2) the above-prepared solutions of
dextrose, (A), (B), (C), and (D) were added in individual flasks to a final
concentration of 1% w/v.
To investigate the PE of the polysaccharides (A, B, C and D), the
individual flasks were inoculated with the probiotic organism
Lactobacillus acidophillus and enteric organism Escherichia coli and in-
cubated at 37 °C for 24 h 2 ml of the culture from each flask were drawn
periodically to measure the optical density (OD) at 600 nm using
thermo scientific evolution 201 UV–Visible spectrophotometer. 0.5 mL
of culture drawn at 0th hour and 24th hour was serially diluted in saline
2
LWT - Food Science and Technology 122 (2020) 109002
and the resultant viable colonies were counted by spread plate method
after 24 h of incubation at 37 °C.
Prebiotic Activity Score (PAS) indicates the capacity of a given
substrate to promote the growth of probiotic organisms exclusively
(Huebner, Wehling, & Hutkins, 2007). PAS was calculated as
Prebiotic Activity Score (PAS)
cfu
Probiotic log ml
on the prebiotic at (24 h− 0h)
=
cfu
Probiotic log ml  on the glucose at (24 h− 0h)
cfu
Enteric log ml on the prebiotic at (24 h− 0h)
−
cfu
Enteric log ml on the glucose at (24 h− 0h)
2.3. Preparation of standard and prebiotic bread
The bread dough was prepared based on typical bread formulation
(Giannou, Kessoglou, & Tzia, 2003). The experiment was carried out in
three different bread varieties Standard Wheat flour Bread (SWB),
Standard Refined flour Bread (SRB), Standard Blended flour Bread
(SBB) (Wheat flour: Refined wheat flour (1:1)). Enrichment by partial
replacement of sugar with pullulan at 2%, 4%, 6%, 8% and 10% was
done in all three varieties of standard bread (SWB, SRB, and SBB) and
the physico-chemical properties of the breads were analyzed.
Bread making was carried out by Standard Sponge and Dough
method(Giannou et al., 2003). After leavening agent preparation, all
the ingredients were mixed together in Taurus® planetary tabletop
mixer (Taurus, Spain). Fat was added at the end to avoid gluten net-
work formation and yeast growth suppression. After first proofing at
40 °C for 1 h the size of dough doubled because of CO2 formed by yeast
fermentation and held inside by gluten network. Later the dough was
kneaded well for even distribution of CO2 and second proofing was
done in a pan for 4 h. After second proofing, the dough was baked at a
temperature of 220 °C for 35 min.
2.4. Characterization of prebiotic bread
2.4.1. Proximate analysis
The proximate analysis of the pullulan polysaccharide and the
pullulan incorporated bread samples were carried out to determine the
moisture content, ash content crude protein and crude fat by standard
methods(AOAC, 2000). The crude fibre was estimated by acid and al-
kali (2.5 N) digestion, 5g of the defatted sample was digested with acid
and alkali followed by incineration in a muffle furnace. The final weight
& Kunkulberga, 2014). The total carbohydrate was calculated by the
difference method.
2.4.2. Thermogravimetric analysis (TGA)
Dynamic TGA tests were carried out using TGA Q50 V2.13 Bulk 39
equipment. 1.66 mg of samples were weighed in alumina pans (70 μl)
and was heated from 30 °C to 700 °C at a rate of 10 °C min −1 under a
nitrogen atmosphere with a flow rate of 30 mL min−1(Haghi &
Carvajal-Millan, 2005).
2.4.3. Colour, texture and surface analysis
The colour index of the prebiotic enriched breads and standard
bread were determined using a Hunter Lab colour spectrophotometer
(Ultra scan VIS, Hunter Lab, Reston, Virginia, USA) and hunter colour
scale. The lightness [L* 0 (black)-100 (white)] and chromaticity para-
meters [a* (red-green)] and [b* (blue/yellow-blue)] were measured.
The measurements were conducted under constant lighting conditions
using reflectance mode at room temperature (25 ± 2 °C) with white
tile as control (L* = 98.76, a* = 0.04, b* = 2.01). All samples were
placed in the sample holder and the reflectance was auto-recorded.
Textural characteristics of the samples and the standard were
S. NithyaBalaSundari, et al.
analyzed using a TA.XT plus Texture Analyzer (Stable Microsystems,
Godalming, Surrey GU7 1 YL, UK). The force applied to the centre of
the sample and firmness of the sample was measured using a 36 mm
cylinder radius probe. The total extensibility and maximum resistance
offered by sample to the extension were measured by kieffer dough and
gluten extensibility rig probe. To evaluate extensibility, 0.4g sample
was clamped at a distance of 18 mm apart and the extensibility hooks
with 1.2 and 4.55 mm diameter were used(Dunnewind, Sliwinski,
Grolle, & Van Vliet, 2003).
The surface morphology and the microstructure of the Pullulan
powder and Bread were recorded using a Scanning Electron Microscope
(VEGA 3 TESCAN, with an integrated EDAX system) at a low-intensity
beam (100 kV) to avoid sample degradation. Specimens were sputtered
with a 16 μg gold layer, and the images were obtained at 1 kV, spot size
4.1 and x100 and ×1000 magnifications.
Additionally, In vitro digestibility, fermentation and PAS of the
bread formulations were done with the procedures described in section
2.2. Characterization of microbial polysaccharides.
2.5. Sensory analysis
The sensory evaluation of the breads was recorded using a nine-
point hedonic scale. The samples were sliced into equal sizes and were
served for evaluation in coded randomized duplicates. The sensory
analysis was performed with 25 semi-trained panellists comprising of
the staffs and the postgraduate students of Centre for Food Technology,
Anna University, Chennai. The panellists were familiar with the sensory
attributes of the breads. An introductory note on the sensory char-
acteristics of bread and the guidelines and instruction for sensory
evaluation using a nine-point hedonic scale was also demonstrated.
Various sensory attributes of the breads were recorded.
2.6. Statistical analysis
In this experiment, all analyses were carried out in triplicates and
the results were reported as mean ± standard deviation. Statistical
analysis was performed using SPSS version 25. The data obtained were
analyzed using univariate Duncan's multiple range test and significant
differences were tested using (P < 0.05).
3. Result and discussion
3.1. In vitro digestibility and fermentation characteristics of microbial
polysaccharide
Resistance to human gastric juice is a significant quality of a pre-
biotic. The DOH of the polysaccharides were evaluated in AHGJ con-
dition (Fig. 1). The DOH of pullulan, gellan, and xanthan were nearly
1.28, 2.96 and 2.19 times higher than that of inulin. DOH of pullulan,
and inulin had a negative correlation with pH and positive correlation
with incubation time. The DOH of pullulan at pH 3 and pH 4 after 6 h
were 0.68 and 0.8 times lower than at pH 2.This is attributed to lower
DOH of glycosidic bonds at higher pH. Resistance to hydrolysis were in
the order inulin (97%) > pullulan (96%) > xanthan (93%) > gellan
(91%) respectively. Hence, foods formulated with these poly-
saccharides reach the intestine to deliver at least 90% of their initial
concentration to the intestinal micro population. Of all three test
samples, pullulan showed better results that are in line with results of
polysaccharides obtained from Gigantochloa levis shoots, Mangifera pa-
jang and rapeseeds (Azmi, Mustafa, Hashim, & Manap, 2012; Al-Sheraji
et al., 2012; X.; Wang et al., 2015).
Selective enumeration of the probiotic organism by prebiotics was
facilitated by lactic acid bacteria (LAB) fermentation. The OD of the
fermentation media demonstrates the growth stimulation of LAB by
different polysaccharides. The stimulation potential is in the following
order: Inulin > Dextrose > pullulan > gellan > xanthan. At the end of
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LWT - Food Science and Technology 122 (2020) 109002
24th-hour dextrose contributed to 66.67% of the maximum cell density
followed by pullulan 61%, gellan 35.8% and xanthan 10.25%
(Supplementary file, Fig. 1a). The sudden increase in the growth pat-
tern of dextrose, xanthan and pullulan after 16h indicates the initiation
of the log phase. Contrastingly, xanthan and gellan expressed only
linear growth patterns after 16 h fermentation. This is attributed to the
insufficient utilization of substrate by the probiotic organism.
The PE of polysaccharides was examined by recording the pH of the
fermentation media with respect to time. pH and growth of LAB were
interrelated as seen clearly in the growth pattern of LAB in the fer-
mentation media. During the initial period of fermentation, all sub-
strates showed a slight decrease in the pH (Supplementary file, Fig. 1b).
After 8h, a significant difference in pH for the pullulan and dextrose
was observed. As fermentation time increased, the pH of the media
decreased at a faster rate indicating LAB growth. After 24h of fermen-
tation, the pH of the pullulan media reduced almost twice. The degree
of decreases in pH was higher for pullulan than all other substrates. This
behaviour was similar to plant-based Fructo-oligosaccharides experi-
ments as reported by (Shalini et al., 2017). Reports dealing with plant-
based polysaccharide are abundant, however microbial polysaccharide
as a prebiotic substrate is a fresh attempt.
The significant difference in acidity level of the media, growth of
LAB and a simultaneous decrease in pH was very high for pullulan and
was not comparable to the case of commercial prebiotic inulin thus
indicating it to be an alternate and excellent prebiotic source.Prebiotic
utilization by probiotics was additionally studied using the viable cell
count. Cell count was higher for pullulan than xanthan and gellan.The
commercial prebiotic inulin and dextrose were utilized by probiotic
strain showing high cell count number than any other substrates. LAB
cell count in pullulan was also similar to inulin and dextrose. E.coli cell
count was very low when compared to other substrates displaying the
selective stimulation of the growth of the probiotic organism. PAS for
the polysaccharides is shown in Fig. 2.
In this study, the inulin PAS was evaluated as 0.59 ± 0.03. Various
PAS scores for inulin was reported (Rubel, Pérez, Genovese, &
Manrique, 2014). reported 0.18 (Shalini et al., 2017), reported
0.21–0.6. This difference may be due to inulin source, cultivar types,
method of extraction and carbon chain length. The relative PAS score of
polysaccharides with inulin as reference was 62.7%, 42.4%, and 33.8%
for pullulan, gellan and xanthan. According to(Rubel et al., 2014),
higher PAS signifies increased probiotic growth and decreased pa-
thogen growth. Higher PAS of pullulan indicates the ability of pullulan
in the selective enumeration of probiotic growth. The PAS derived ex-
perimentally are in accordance with the prebiotic properties and PAS of
plant-based polysaccharides (Shalini et al., 2017; Zhang et al., 2013).
3.2. Prebiotic bread
The competence of pullulan among the polysaccharides to serve as a
potential prebiotic was validated in the above studies. Therefore pull-
ulan was used as sole prebiotic for all bread formulations for further
examination. Three different SB varieties namely SWB, SRB, SBB were
prepared. Initially, the pullulan enrichment concentration was opti-
mized in SRB by varying the concentration of pullulan as 2%, 4%, 6%,
8%, 10%. Based on the overall acceptance in sensory analysis 2% en-
richment was preferred for further examination in SWB and SBB.
3.3. Proximate analysis
Proximate characters such as moisture content, mineral content,
protein content, crude fibre and carbohydrate content were analyzed
for standard and 2% pullulan added PEB samples (Table 1).
The proximate composition of pullulan enriched bread revealed that
the moisture content of PEB was lesser than SB signifying enhanced
water binding. The crude fibre content of PEB increased by three-fold
when compared to the SB. A seven to nine-fold increase in total fibre in
S. NithyaBalaSundari, et al.
Fig. 1. Degree of hydrolysis (%) of the polysaccharides – Inulin (♦), Pullulan (▲),Gellan (■) and Xanthan ( × ) at (a) pH 2, (b) pH 3, (c) pH 4.
bakery product with partial replacement of sucrose by fructooligo-
saccharides (40%, 60%, and 80%) was reported (Handa, Goomer, &
Siddhu, 2012) however, the other constituents like protein, fat, carbo-
hydrate and ash levels remained the same.
3.4. Thermogravimetric analysis (TGA)
TGA curves demonstrating the thermal properties of pullulan was
studied (Supplementary file, Fig. 2). Pullulan degraded above 265 °C
thus implying pullulan can withstand the baking temperature. In the
thermograph of pullulan, the first thermal event was a weight loss in
the range of 250–350 °C, which was related to water evaporation in the
sample. Evaporation occurs at the liquid/air interface, and the water is
readily available at the surface. The second thermal event started from
4
LWT - Food Science and Technology 122 (2020) 109002
350 to 650 °C and was related to depolymerisation of the poly-
saccharide chains(Grabowska, Malinowski, Szucki,& Byczyn'ski, 2016)
3.5. Colour, texture and microstructure analysis
The colour values for refined wheat flour bread containing 5 dif-
ferent prebiotic concentrations were examined. Bread enriched with 2%
pullulan shared similar colour characteristics with standard bread
(Supplementary file, Fig. 3).
Colour values for SB and PEB are shown in Table 2. As the reducing
sugars caramelize during baking PEB were slightly darker than SB
(Whistler & BeMiller, 2012).The development of colour in bread is the
result of simultaneously the Maillard and caramelization reactions
(Zanoni, Peri, & Bruno, 1995). 2% PEB did not deviate much from the
S. NithyaBalaSundari, et al. LWT - Food Science and Technology 122 (2020) 109002

Fig. 2. Prebiotic activity score of the polysaccharides.

All the obtained data were subjected to univariate with Duncan's multiple range
test. Same variables above the bars are not significantly different at 0.05 LSD.

SB and hence was found most acceptable whereas a further increase in

pullulan concentration reduced the visual acceptability of breads.
Texture differences were observed with respect to baked product
quality Fig. 3. 2% PEB had texture quality as good as the SB. When
pullulan was enriched, the peak force required to penetrate bread tends
to decline indicating enhanced softness of PEB. The SB showed higher
firmness signifying its hardness.
The morphology of the pullulan and bread samples were analyzed
by SEM to observe the surface morphology and the homogeneity of the
sample (supplementary file, Fig. 4 & Fig. 5). SEM micrographs of the SB
dough and PEB dough clearly indicated the gluten network interference
by pullulan in the prebiotic formulations. The smooth surface was ob-
served in all the bread samples indicating mixture homogeneity. This is
further supported by the compact cross-section morphology images
indicating a strong interaction between the polysaccharides. These re- Fig. 3. Texture chart for pullulan enriched bread samples. The texture prop-
sults were in agreement with studies on other gums reported (J. Wang, erties of bread crumb = and bread crust = were evaluated. (a)
De Wit, Boom, & Schutyser, 2015). prebiotic enrichment in refined wheat flour bread at five different prebiotic
concentration (b) prebiotic enrichment in six different bread varieties at 2%
prebiotic concentration. Standard Refined wheat flour Bread (SRB), Prebiotic
3.6. In vitro digestibility test Refined wheat flour Bread (PRB), Standard Wheat flour Bread (SWB), Prebiotic
Wheat flour Bread (PWB), Standard Blended Bread (SBB), Prebiotic Blended
Bread (PBB). Mean ± Standard Deviation (SD) values of triplicates.
The hydrolysis of prebiotic formulations by AHGJ against time re-
veals the prebiotic characteristics of the formulations developed
(Fig. 4a). 3.7. In vitro fermentation and PAS of pullulan enriched bread
The DOH of all the samples during the initial period of digestion was
gradual and as time increases the DOH also increases for all the sam- LAB and E.coli cell count (log CFU/mL) on different carbon sources
ples. Compared with SB, the PEB expressed lower DOH which is in were studied. E.coli cell count for both pullulan gum and pullulan en-
agreement with the hydrolytic behaviour of pullulan in AHGJ en- riched bread sample was lower than LAB cell count value.
vironment. The difference in DOH of formulated breads from SB is Dextrose showed higher LAB count followed by inulin and pullulan
purely attributed to the presence of pullulan. Hence, it is evident that a in order. This is due to the fact that carbohydrates with shorter chain
minimum of 90% of the pullulan in the bread formulation will be lengths were fermented at a faster rate. But cell count in pullulan was
available for probiotics inhabiting the intestine. more or less similar to commercial prebiotics inulin thereby exposing its
possibility of stimulating the growth of probiotic organism at a faster
rate. The cell count of PEB was much higher than SB. The higher colony

Table 1
Proximate analysis of the bread formulations per 100g.
Sample Moisture (g) Ash (g) Protein (g) Fat (g) Crude Fiber (g) Carbohydrate (g)

c b a c a
SRB 24.34 ± 0.41 1.54 ± 0.23 0.12 ± 0.05 7.87 ± 0.61 0.00 ± 0.00 66.13 ± 0.22c
PRB 22.14 ± 0.65ab 1.71 ± 0.25d 0.75 ± 0.10b 1.93 ± 0.55a 0.11 ± 0.02ab 73.36 ± 0.17f
SWB 22.28 ± 0.33ab 1.61 ± 0.18c 1.18 ± 0.17cd 5.32 ± 0.37b 0.26 ± 0.14b 69.35 ± 0.17e
PWB 22.48 ± 0.56b 1.36 ± 0.19a 1.24 ± 0.04d 6.23 ± 1.18b 0.69 ± 0.15c 68.00 ± 0.34d
SBB 27.17 ± 1.43d 1.73 ± 0.28e 1.13 ± 0.06cd 7.9 ± 1.40c 0.15 ± 0.12ab 61.92 ± 0.29a
PBB 20.83 ± 0.84a 1.76 ± 0.32f 1.04 ± 0.14c 12.78 ± 0.69d 0.95 ± 0.23d 62.64 ± 0.31b

Standard Refined wheat flour Bread (SRB), Prebiotic Refined wheat flour Bread (PRB), Standard Wheat flour Bread (SWB), Prebiotic Wheat flour Bread (PWB),
Standard Blended Bread (SBB), Prebiotic Blended Bread (PBB). Mean ± Standard Deviation (SD) values of triplicates. All the obtained data were subjected to
univariate with Duncan's multiple range test. Values followed by the same superscripts are not significantly different at 0.05 LSD.

5
S. NithyaBalaSundari, et al. LWT - Food Science and Technology 122 (2020) 109002

Table 2
Colour analysis of 2% pullulan incorporated breads (PRB, PWB, and PBB) and standard breads (SRB, SWB, and SBB). The colour index of the breads was examined
with SRB as standard.
ΔE Δ L* Δ a* Δ b*

PRB 03.39 ± 0.19a −03.26 ± 0.23e 0.05 ± 0.07a −0.87 ± 0.28b

SWB 10.95 ± 0.51b −10.10 ± 0.54d 03.51 ± 0.34c 02.27 ± 0.70c
PWB 22.07 ± 0.40d −19.76 ± 0.47b 06.59 ± 0.02d 07.26 ± 0.32e
SBB 29.02 ± 0.24e −26.85 ± 0.15a −0.03 ± 0.08a −11.00 ± 0.19a
PBB 12.39 ± 0.17c −10.94 ± 0.29c 03.09 ± 0.01b 04.94 ± 0.21d

Standard Refined wheat flour Bread (SRB), Prebiotic Refined wheat flour Bread (PRB), Standard Wheat flour Bread (SWB), Prebiotic Wheat flour Bread (PWB),
Standard Blended Bread (SBB), Prebiotic Blended Bread (PBB). ΔL* = (L* sample - L* standard) = difference in lightness and darkness (+= lighter, - = darker), Δa*
= (a* sample - a* standard) = difference in red and green (+= redder, - = greener), Δb*= (b* sample - b* standard) = difference in yellow and blue (+=
yellower, - = bluer). Mean ± Standard Deviation (SD) values of triplicates. All the obtained data were subjected to univariate with Duncan's multiple range test.
Values followed by the same superscripts are not significantly different at 0.05 LSD.

3.8. Sensory analysis

The sensory scores obtained with respect to various quality attri-

butes were analyzed and presented in Fig. 5.
The external & internal sensorial characters of PEB at five different
level (2%, 4%, 6%, 8%, 10%) with standard bread, bread at 2% en-
richment level resulted in overall better acceptance. Hence further
analysis was carried out with 2% Prebiotic enriched Refined wheat
flour Bread (PRB), Wheat flour Bread (PWB), Blended flour Bread
(PBB). In six cases the prebiotic enriched bread varieties havea higher
sensory score, the overall acceptability of all prebiotic enrichment at
2% has a higher value than standard bread.

4. Conclusion

Microbial polysaccharides showed significant prebiotic activity. The

performance of pullulan was observed to be notable. In vitro digest-
ibility and In vitro fermentation studies revealed that pullulan exhibited
a higher degree of resistance to AHGJ and better compatibility as a
carbon source to the probiotic organism (LAB) than xanthan and gellan.
Higher PAS of pullulan indicates its selective stimulation of probiotic
organism growth and suppression of pathogenic organism growth
thereby satisfying the ideal requirements for an efficient prebiotic. In
covenant with the above results, PEB showed more resistance to gastric
acid than SB. The suitability of the PEB and SB as a carbon source for
the probiotic organism was examined was found that the PEB showed
higher compatibility to probiotic organisms and was also confirmed
with higher PAS for PEB than SB. With all the above-discussed qualities
of pullulan, it is evident that pullulan can be a potential prebiotic with
versatile significance. However, this is a preliminary comparative study
Fig. 4. Degree of hydrolysis (DOH) and Prebiotic Activity Scores (PAS) of of the prebiotic property of PEB. Further studies on other physico-
Pullulan enriched Bread samples. Standard Refined wheat flour Bread chemical, organoleptic properties and in vivo studies can aid in evi-
(SRB) = , Prebiotic Refined wheat flour Bread (PRB) = , Standard dencing pullulan as a potential prebiotic.
Wheat flour Bread (SWB) = , Prebiotic Wheat flour Bread
(PWB) = , Standard Blended Bread (SBB) = , Prebiotic Blended Author contributions
Bread (PBB) = . Mean ± Standard Deviation (SD) values of triplicates.
(a) Degree of hydrolysis (DOH) of PEB. (b) Prebiotic Activity Scores (PAS) of
Nithya Bala Sundari S: Methodology and experimentation
PEB. All the obtained data were subjected to univariate with Duncan's multiple Nivedita V: Design of experiments Nandhini Devi G and Usha
range test. Same variables above the bars are not significantly different at 0.05 Antony: Conceptualization, validation of Experimental designs and
LSD. Supervision Chakravarthy M and Srisowmeya G: Validation, data
curation, writing and original draft preparation.
count was also achieved in PRB and PBB. However, PWB showed lower
cell count than SWB. This indicates the decrease in pullulan activity Declaration of competing interest
when mixed with wheat flour however in the case of wheat and refined
wheat flour blends the PAS values of these bread samples were still The authors declare no conflict of interest.
satisfactory (Fig. 4b).
Appendix A. Supplementary data

Supplementary data to this article can be found online at https://

doi.org/10.1016/j.lwt.2019.109002.

6
S. NithyaBalaSundari, et al.
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