Applied Food Research 2 (2022) 100136

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Applied Food Research

journal homepage: www.elsevier.com/locate/afres

Inulin as a prebiotic for the growth of vegan yoghurt culture in pea

protein-based vegan yoghurt-ice cream, while improving the textural
properties
Vikram Reddy Narala a,b,∗, Madara Asnate Jugbarde a, Ivars Orlovs a, Markus Masin b
a
Nature Foods Ltd, Ģertrūdes iela 53 - 7, Rīga LV-1011, Latvia
b
Medical Institute for Nutrition Science and Technology, Uriekstes iela 1, Riga LV-1005, Latvia

a r t i c l e i n f o a b s t r a c t

Keywords: The study aimed to develop a vegan yoghurt-ice cream made up of pea protein isolate with inulin as a prebiotic
Pea proteins for the growth of yogurt culture while investigating the influence of inulin on textural properties – hardness,
Ice cream melting, overrun, and colour. Freeze-dried cultures were added at 0.02% (w/v) to the mixes made up of 0, 1, 2,
S. thermophilus
3, and 4% inulin, and incubation was done at 45 °C until it reaches a pH of 4.5. A significant (p<0.05) increase
L. bulgaricus
in counts of Streptococcus thermophilus and Lactobacillus bulgaricus were observed with the addition of inulin (2,
Inulin
Prebiotic 3, and 4%) and count was maintained more than 6 log CFU/g during 10 days of storage. The maximum overrun
of 42.87 ± 3.74% was obtained with the addition of 4% inulin. The fermentation of pea protein ice cream mixes
showed a significant (p<0.05) reduction in the hardness and melting times increased with the inulin addition. The
colour values L∗ , a∗ and b∗ were influenced by fermentation and blueberry-juice concentration in the formulation.
It can be concluded that the addition of inulin to the pea protein yoghurt-ice cream showed prebiotic activity
and influenced the textural properties during the development of vegan probiotic food as a dairy alternative.

1. Introduction isolates in the production of plant-based dairy replacements (Ge et al.,

Veganism, which involves avoiding the use of all animal products,
including meat, dairy products, honey, eggs, and other items, has ex-
ploded in popularity around the world in recent years (Cramer et al.,
2017). Veganism is practised for a variety of reasons, including moral,
ethical, animal welfare, dietary limitations, and the desire to enhance
one’s health, with a focus on health and ethics (Dyett et al., 2013; Fox &
Ward, 2008; Ruby, 2012). In this case, plant protein-based dairy alterna-
tives are useful for addressing allergies, lactose intolerance, and vegan
diets (Chen et al., 2019; Cramer et al., 2017; Wangcharoen, 2012).
Pea protein isolate is the most cost-effective and long-lasting option
for generating dairy substitutes since it has the best functional and nu-
tritional qualities while containing the fewest allergies (Lu et al., 2020).
Pea has 65 to 80 percent globulin proteins, 10 to 20 percent albumin
proteins, and a high lysine content (Guler-Akin et al., 2021). Pea pro-
tein (mostly pea protein hydrolysates and particular peptide fractions)
contains antioxidant, antihypertensive, anti-inflammatory, cholesterol-
lowering, and bacterial modulation properties (Li & Aluko, 2010;
Liao et al., 2019). Furthermore, functional traits such as foaming, emul-
sification, oil-holding capacity, water-holding capacity, solubility, and
food texturing are the most attractive features for using pea protein
2020; Guler-Akin et al., 2021; Liao et al., 2019).
As a result, the creation of pea protein-based yoghurt-ice cream
could be an appealing dairy ice cream alternative to address the grow-
ing need for products designed for certain diets, containing ingredients
with nutritional and physiological benefits and containing least aller-
gens (Aboulfazli et al., 2016; Crizel et al., 2014; Cruz et al., 2009).
Though plant-based materials are suitable for the growth of probiotic
cultures in the development of fermented products such as yoghurt, cul-
tured food products are only considered probiotics if they contain a
minimum viable bacterial count of 6 log CFU/g (Konar et al., 2018),
which can be obtained by consuming 100 g or 100 mL of food on
a daily basis (Aydar et al., 2021; Homayouni et al., 2020). As a re-
sult, yoghurt ice cream development hinges on the viability of health-
promoting bacteria during manufacturing and storage. As a response,
inulin has been used as a prebiotic in the past to preserve the viability
of Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus aci-
dophilus, Bifidobacterium, and other bacteria. Supplementing with inulin
not only preserves the activity and survivability of some L. casei and L.
acidophilus strains, but it also drastically reduces the generation time of
Streptococcus and Lactobacillus (Oliveira et al., 2011a, 2011b). Accord-
ing to studies by Isik et al. (2011) and Łopusiewicz et al. (2020), inulin

∗
Corresponding author at: Nature Foods Ltd, Ģertrūdes iela 53 - 7, Rīga LV-1011, Latvia.
E-mail address: vikramreddynarala@gmail.com (V.R. Narala).

https://doi.org/10.1016/j.afres.2022.100136
Received 14 April 2022; Received in revised form 20 May 2022; Accepted 24 May 2022
Available online 26 May 2022
2772-5022/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
V.R. Narala, M.A. Jugbarde, I. Orlovs et al. Applied Food Research 2 (2022) 100136

Table 1
Formulations of unfermented and fermented pea protein ice creams.

Ice cream mix formulations

Ingredients
A B C D E F

Pea protein 5 5 5 5 5 5
isolate (w/v%)
Sugar (w/v%) 20 20 20 20 20 20
Rapeseed oil 10 10 10 10 10 10
(v/v%)
Stabilizing 0.7 0.7 0.7 0.7 0.7 0.7
mixture
EXCELAIS 602
(w/v%)
Blueberry juice 2 2 2 2 2 2
concentrate
(v/v%)
Dextrose (v/v%) 1 1 1 1 1 1
Potassium 0.2 0.2 0.2 0.2 0.2 0.2
tripolyphos-
phate
(w/v%)
Bacterial 0 0.02 0.02 0.02 0.02 0.02
cultures (w/v%)
Inulin (w/v%) 0 0 1 2 3 4
Water (v/v%) 61.1 61 60 59 58 57

increased the count of S. thermophilus and L. bulgaricus and aided in
bacterial activity maintenance. Furthermore, using inulin in ice cream
has improved the ice cream’s textural properties (Pintor et al., 2017;
and Samakradhamrongthai et al., 2021). Most of the studies were done
on conventional dairy based yoghurt and yoghurt ice creams, as far as
known, no previous studies were made on finding inulin prebiotic ac-
tivity in pea protein based vegan yoghurt-ice cream. Hence, the goal of
this study was to develop a pea protein yoghurt-ice cream with inulin
supplementation as a dairy alternative while investigating the role of
inulin as a prebiotic in the growth of S. thermophilus and L. bulgaricus,
as well as its impact on the textural properties of the vegan yoghurt-ice
cream.
2. Materials and methods
culture. All the inoculated mixes were incubated in ICN 55 natural con-
vection incubator (AGROLAB, Italy) at 45 °C until pH reduced to 4.5 and
cooled to 20 °C using an ice-filled sink for immediate freezing. Freezing
of all unfermented and fermented mixes was done at -12 ± 1 °C temper-
ature using batch type Frigomat G10 ice cream freezer (Frigomat, Italy)
for 7 min and filled in 100 mL sampling cups for 1st, 5th and 10th day
storage in a hardening chamber (Midea, China) at -18 ± 1 °C for further
analysis.
Note: The mixes B, C, D, E, and F were added with only 5% of sugar
before fermentation in the view of bacterial growth and the remaining
15% was added after incubation followed by freezing, but in the case
of unfermented mix A, whole 20% sugar was added immediately before
homogenisation.

2.3. pH measurements
2.1. Materials

The pH value of ice cream mixes was measured using an InLab®

Pea protein isolates (79% of protein content) (Danisco, Denmark),
Expert Pro-ISM pH electrode (Mettler Toledo, Switzerland).
locally sourced sugar (AB ‘Nordic Sugar Kedainiai’, Lithuania) and rape-
seed oil (AS ‘Scanola Baltic’, Estonia), Blueberry juice concentrate (SIA
‘Puratos’, Latvia), stabilizer mix Excelais 602 (Mono- and diglycerides of
2.4. Microbiological analysis
fatty acids, Guar gum, Locust bean gum, Carrageenan) (Rikevita SDN.
BHD, Malaysia), Orafti HPX inulin (Beneo, Belgium), Direct vat inoc-
The counts of S. thermophilus were enumerated on M17 agar and
ulation freeze-dried vegan yoghurt culture (Streptococcus thermophilus,
incubated at 37 °C for 48 h under aerobic conditions. Enumeration of L.
Lactobacillus delbrueckii subsp. Bulgaricus) (Danisco, France), Potassium
bulgaricus counts was made on MRS agar and incubated aerobically at
tripolyphosphate (Omnisal Gmbh, Germany), Dextrose (Cargill, UK) and
37 °C for 72 h (Dave & Shah, 1996). The results were recorded as the
water were used as ingredients for ice-cream production.
log number of colony-forming units per gram (CFU/g).

2.2. Pea protein yoghurt-ice cream preparation

2.5. Overrun
The formulations developed for the pea protein yoghurt-ice cream
mixes to employ inulin as a prebiotic are presented in Table 1. All the Overrun was measured by comparing the weight of the ice cream
ice-cream mixes were made by homogenizing the ingredients in wa- mix and ice cream in a fixed volume (100 mL) container, referring to
ter at 3000 rpm for 5 min using IKA® T18 digital ULTRA TURRAX® the method mentioned in (Muse & Hartel, 2004). The average value of
high shear mixer (IKA®, Germany) and followed by pasteurisation on ten overrun measurements in percentage was taken per sample using
GuardianTM 7000 magnetic stirrer (Ohaus®, USA) speed of 900 rpm the following equation:
with heating plate at 85 °C for 10 min. The unfermented mix (A) was
immediately cooled to 20 °C using the ice-filled sink and aged at 4 °C 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑖𝑐𝑒 𝑐𝑟𝑒𝑎𝑚 𝑝𝑟𝑒𝑚𝑖𝑥 − 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑖𝑐𝑒 𝑐𝑟𝑒𝑎𝑚
% 𝑜𝑓 𝑜𝑣𝑒𝑟𝑟𝑢𝑛 = × 100
temperature around 2 h to hydrate all the ingredients. The fermented 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑖𝑐 𝑒𝑐 𝑟𝑒𝑎𝑚
mixes (B, C, D, E, and F) were cooled to 45 °C immediately after pas- (1)
teurisation and inoculated with 0.02% (w/v) freeze-dried vegan yogurt

2
V.R. Narala, M.A. Jugbarde, I. Orlovs et al.
Table 2
Change in pH value of the pea protein yoghurt-ice cream mixes (B – 0%, C – 1%, D –
2%, E – 3%, and F – 4% inulin) during incubation.
Time B C
0h 5.69 ± 0.07a 5.71 ± 0.05a
1h 5.47 ± 0.11a 5.45 ± 0.13ac
2h 5.37 ± 0.09a 5.38 ± 0.12a
3h 5.02 ± 0.1a 4.96 ± 0.09a
4h 4.65 ± 0.05a 4.63 ± 0.04a
∗
Different superscript letters (a,b,c) show significant differences (p<0.05) in a row.
2.6. Melting rate
The melting of 1st, 5th and 10th day stored (-18 ± 1 °C) ice cream
samples were measured by placing 100 mL of ice cream on 1 mm stain-
less steel mesh at ambient temperature (20 ± 1 °C), and the weight of
the melted ice cream was noted every 10 min (Pintor et al., 2017). The
plot diagram was developed using the percentage of melted ice cream
versus the time. Average of triplicate values were taken.
2.7. Textural analysis
Textural analysis was conducted at ambient temperature (20 ± 1 °C)
using TA.HDplus Texture Analyser (Stable Micro Systems Ltd, England)
equipped with 2.5 cm diameter stainless steel cylindrical probe. The
frozen sample stored at -18 ± 1 °C was removed from the freezer shortly
before the analysis. The texture analysis conditions were as follows: pen-
etration depth, 15 mm; trigger force, 5.0 g; probe speed during penetra-
tion, 3.3 mm/s; probe speed pre and post penetration, 3.0 mm/s. The
probe was penetrated one place on the largest smooth surfaces of the
ice cream samples, which remained in the plastic cup and six measure-
ments were recorded for each sample. The hardness of 1st, 5th, and 10th
day-stored samples were calculated as peak force (N) during penetration
(Akalın et al., 2008).
2.8. Colour measurements
The colour of one day-stored pea protein ice creams was measured
using ColorTech-PCM metre (Accuracy Microsensors, USA) with “Col-
orsoftQCW” software. An average value of ten measurements was taken
per sample. The L∗ , a∗ , b∗ colour space was used to determine the colour
of the ice cream, where L∗ gives the lightness, +a∗ means the red direc-
tion, -a∗ means the green direction, +b∗ means the yellow direction, -b∗
means the blue direction.
2.9. Data processing
Data analysis and acquisition were completed using Microsoft Office
Excel v16.0 and SPSS 19.0 software. Analysis of variance (ANOVA) and
Tukey’s test was used to compare means, differences were considered
significant at a level of p<0.05.
3. Results and discussion
3.1. pH shifts and bacterial growth in vegan yoghurt-ice cream
Because the yoghurt cultures Streptococcus thermophilus and Lacto-
bacillus bulgaricus can produce acid, the pH of all the fermented pea
protein ice cream mixes was decreased during the incubation period, as
indicated in Table 2 (Akın et al., 2007; Oliveira et al., 2009). After 4 h of
incubation, the pH of the mixes D, E, and F with the greater amounts of
2, 3, and 4% inulin was significantly lower (p<0.05) than the remaining
two mixes B and C with 0 and 1% inulin. These findings can be explained
by the fact that inulin’s prebiotic nature causes bacteria to grow faster
and produce more acid, lowering pH levels (M. B. Akın et al., 2007;
Applied Food Research 2 (2022) 100136
D E F
5.71 ± 0.03a 5.73 ± 0.02a 5.72 ± 0.06a
5.43 ± 0.02b 5.45 ± 0.08c 5.44 ± 0.05c
5.22 ± 0.08b 5.15 ± 0.05c 5.12 ± 0.04c
4.72 ± 0.14b 4.69 ± 0.03bc 4.67 ± 0.07c
4.51 ± 0.11b 4.46 ± 0.07bc 4.44 ± 0.03c
Oliveira et al., 2009). According to Guggisberg et al., 2009, an increase
in inulin level during yoghurt manufacturing reduces incubation time.
Inulin supplementation not only has a conservation effect on the activ-
ity and viability of some L. casei and L. acidophilus strains, but it also
significantly reduces the generation time of Streptococcus thermophilus
and L. bulgaricus, according to Oliveira et al. (2011a, 2011b).
Due to the symbiotic growth features of yoghurt bacteria, Streptococ-
cus thermophilus resulted in a higher count from 8.33 to 9.23 log CFU/g
after 4 h incubation of mixes (B, C, D, E, and F) compared to Lacto-
bacillus bulgaricus count from 7.97 to 9.14 log CFU/g as shown in Figs.
1 and 2 (Sarkar, 2008). Since the prebiotic nature of inulin stimulated
the growth of yoghurt bacteria, the addition of inulin to mixes D, E,
and F (2, 3, and 4%) led to a significant (p<0.05) increase in counts
of both bacteria when compared to mix B without inulin. It was dis-
covered that inulin content and bacterial counts have a strong positive
connection (inulin vs S. thermophilus: R2 = 0.9125; inulin vs L. bulgari-
cus: R2 = 0.9117). The research results are line with the observations of
the authors Oliveira et al. (2009, 2011a, 2011b), who investigated the
prebiotic activity of inulin in fermented milk, and Boeni and Pourah-
mad (2012), encountered that adding inulin to yoghurt increases the
viable count of Streptococcus thermophilus and Lactobacillus bulgaricus.
After freezing mixtures B, C, D, E, and F, counts of both bacteria de-
creased by about 1 log CFU/g, irrespective of the amount of inulin con-
tent in the mix. Mechanical stressors, freezing, and incorporating air into
the fermented ice cream injures the probiotic bacterial cells, resulting
in a reduction in count (Davidson et al., 2000; Haynes & Playne, 2002;
Shah & Ravula, 2001; Akın et al., 2007). After the one day of storage at
-18 ± 1 °C, there was no significant difference in counts of both bacte-
ria in all pea protein yoghurt-ice creams compared to just frozen mixes,
however samples B, C, and D showed a gradual drop after 5th and 10th
day storage. The counts of both bacteria did not change in the pea pro-
tein yoghurt-ice creams E and F with the addition of 3% and 4% inulin,
respectively, throughout storage from the first to the tenth day. The find-
ings support those of Akalın et al. (2004), Aryana and Mcgrew (2007),
and Donkor et al., (2007), researchers found that inulin protects yo-
ghurt bacteria by enhancing their survival and activity during storage.
Akın et al. (2007) found that Streptococcus thermophilus and Lactobacillus
bulgaricus were more viable for more than 7 days in yoghurt-ice creams
with 1 and 2% inulin compared to ice creams without inulin. Even after
10 days of storage, the counts of both bacteria were greater than 6 log
CFU/g in all pea protein yoghurt-ice creams (B, C, D, E, and F). Only
cultured food products with a minimum viable bacterial count of 6 log
CFU/g, as determined by daily consumption of 100 g or 100 mL of food
(Aydar et al., 2021; Konar et al., 2018; Homayouni et al., 2020), are
considered probiotics, as defined by a standard requiring a minimum of
6 log CFU/g of S. thermophilus and/or L. bulgaricus (IDF, 1992).
3.2. Melting rate
The initial dripping and complete meltdown times of all pea pro-
tein ice creams (A, B, C, D, E, and F) were more than 40 and
130 min (Table 3), respectively. The authors Guler-Akin et al. (2021);
Loffredi et al. (2021), and da Silva and Lannes (2011) mentioned below
20 and 60 min for initial dripping and complete meltdown of dairy ice
3
V.R. Narala, M.A. Jugbarde, I. Orlovs et al. Applied Food Research 2 (2022) 100136

10

9
B
log CFU/g

8 C
D
7
E

6 F

5
Incubation Freezing 1st day 5th day 10th day

Storage period

Fig. 1. Change in Streptococcus thermophilus count after incubation for 4 h, freezing, and storage for 1st, 5th, and 10th day of pea protein yoghurt-ice creams (B –
0%, C – 1%, D – 2%, E – 3%, and F - 4% inulin).

10

9
B
log CFU/g

8 C
D
7
E
F
6

5
Incubation Freezing 1st day 5th day 10th day

Storage period

Fig. 2. Change in Lactobacillus bulgaricus count after incubation for 4 h, freezing, and storage for 1st, 5th, and 10th day of pea protein yoghurt-ice creams (B – 0%,
C – 1%, D – 2%, E – 3%, and F - 4% inulin).
∗
Different letters (on top of the bars) show significant differences (p<0.05) in hardness during 1st, 5th, and 10th day storage of unfermented and fermented pea
protein ice creams with 0, 1, 2, 3, and 4% inulin content.

creams, respectively. The slower melting of the pea protein ice creams
in the study was due to pea protein isolates capability to form a strong
gel network with water molecules in the formulation (Boye et al., 2010).
Slower meltdown was observed in all the fermented ice creams (B, C,
D, E, and F) compared to unfermented ice cream (A) after 1st, 5th, and
10th day of storage due to lower pH values (around 4.5) can change the
protein networking with the fat, water and other constituents in the ice
cream formulation. An increase in acidity of ice cream mix due to the
addition of lactic acid bacteria leads to denaturing of proteins, reflect-
ing the higher melting resistance of ice cream (Favaro-Trindade et al.,
2007). However, the addition of inulin to ice cream increases the ini-
tial dripping and complete meltdown times due to the water reten-
tion capacity of inulin, leading to immobilisation of water molecules
and less water crystallization in the ice cream (Akalın & Erisir, 2008;
Pintor et al., 2014, 2017). Similar results were shown in this study, the
fermented samples D, E, and F (with 2, 3, and 4% of inulin) produced
higher melting times compared to the fermented sample B without in-
ulin after 1st, 5th and 10th day of storage. The increase in meltdown
times were observed in all unfermented and fermented pea protein ice
creams with an increase in storage times, the results are in agreement
with the studies Ahmad et al. (2020), Akalın et al. (2008), and Guler-
Akin et al. (2021) where, they mentioned the possibility of larger ice
crystal formation during the storage of ice cream.
3.3. Overrun
During this investigation, the overrun of all unfermented and fer-
mented pea protein ice creams was between 35 and 43%. Guler-
Akin et al. (2021) discovered a negative correlation between amount
of pea protein isolate and ice cream overrun values due to very high
viscosity gel-matrix formation, which may negatively affect the incor-
poration of air during the freezing process (Sivasankari et al., 2019). Ac-
cording to Pereira et al. (2011) and Badilli (2020), adding soy protein
extract and chick pea flour to ice cream formulations leads in higher

4
V.R. Narala, M.A. Jugbarde, I. Orlovs et al. Applied Food Research 2 (2022) 100136

Table 3
Change in melting (%) of unfermented (A – 0% inulin) and fermented (B – 0%, C – 1%, D – 2%, E – 3%,
and F - 4% inulin) pea protein ice creams after storage for 1st, 5th, and 10th day.

Storage day Ice cream 10 min 40min 70min 100min 130min

1st A 0 0.89 ± 0.32% 30.87 ± 2.78% 74.76 ± 3.09% 93.94 ± 3.22%

B 0 0.48 ± 0.27% 21.42 ± 2.96% 61.64 ± 1.78% 90.82 ± 1.57%
C 0 0.62 ± 0.31% 20.13 ± 3.55% 60.73 ± 3.01% 90.07 ± 3.21%
D 0 0 18.54 ± 1.93% 58.47 ± 2.50% 88.16 ± 2.11%
E 0 0 17.63 ± 1.57% 56.32 ± 1.54% 88.07 ± 3.05%
F 0 0 16.71 ± 2.30% 55.48 ± 1.32% 86.98 ± 2.45%
5th A 0 0 29.14 ± 2.15% 72.14 ± 3.34% 92.58 ± 1.77%
B 0 0 20.34 ± 2.32% 60.13 ± 2.02% 87.79 ± 2.23%
C 0 0 20.32 ± 3.96% 60.18 ± 2.67% 88.13 ± 3.78%
D 0 0 17.64 ± 1.05% 56.98 ± 2.23% 87.54 ± 1.96%
E 0 0 18.02 ± 1.71% 57.04 ± 1.67% 86.23 ± 2.04%
F 0 0 17.04 ± 2.24% 54.18 ± 2.05% 85.10 ± 3.23%
10th A 0 0 23.15 ± 1.73% 68.20 ± 3.04% 90.82 ± 3.35%
B 0 0 18.94 ± 2.31% 58.17 ± 2.05% 85.32 ± 2.35%
C 0 0 17.64 ± 3.75% 55.07 ± 3.45% 85.63 ± 1.78%
D 0 0 14.13 ± 2.23% 53.11 ± 2.69% 83.42 ± 2.04%
E 0 0 14.27 ± 1.89% 52.36 ± 1.78% 82.87 ± 1.92%
F 0 0 13.97 ± 2.33% 51.50 ± 1.93% 83.01 ± 2.19%

mix viscosities and lower overrun values. Unfermented (A) and fer-
mented (B and C) pea protein ice creams had no significant difference
in overrun (p>0.05). However, compared to the ice creams A, B, and
C, adding 2, 3, and 4% inulin to the fermented ice creams (D, E, and
F) resulted in a significant (p<0.05) increase in overrun. The greater
overrun values in Table 4 can be explained by the fact that adding in-
ulin to the ice cream increases viscosities, which promotes more effi-
cient air incorporation and the development of smaller air cells dur-
ing freezing. The authors Akalın et al. (2008), Pintor et al. (2017), and
Samakradhamrongthai et al. (2021) found that adding inulin between
2 and 4% improved the overrun of dairy ice cream.
Table 4
Change in overrun of unfermented (A – 0% inulin) and fermented (B – 0%,
C – 1%, D – 2%, E – 3%, and F - 4% inulin) pea protein ice creams.
Ice cream mix Overrun (%)
A 35.28 ± 3.58a
B 36.1 ± 4.24a
C 36.46 ± 3.34a
D 39.5 ± 2.78b
E 41.05 ± 3.51bc
F 42.87 ± 3.74c
∗ Differentsuperscript letters (a,b,c) within a column show significant differ-
ences (p<0.05).

3.4. Texture analysis

According to the results shown in Fig. 3, the significant (p<0.05) de-
crease in the hardness of all the fermented pea protein ice creams (B,
C, D, E, and F) was obtained compared to the unfermented ice cream
(A) during 1st, 5th, and 10th day storage. The reduction in hardness can
be resultant of acid-induced gel formation of pea protein isolates with
the networking of oil and other constituents in the mix during bacterial
fermentation. Klost et al. (2019b, 2020) found that the fermentation
induced pea protein gels produce a lower G’ (storage modulus) value
compared to heat induce pea protein gels. The yoghurt-ice creams D, E

500
c
450
400 b
a x
350
A
Hardness (N)

300 y
X Y B
A z q q
250 A
B C Z Q Q C
200 C
D
150 E
100 F
50
0
1st day 5th day 10th day
Storage period

*Different letters (on top of the bars) show significant differences (p<0.05) in hardness during 1st, 5th, and 10th
day storage of unfermented and fermented pea protein ice creams with 0, 1, 2, 3, and 4% inulin content.

Fig. 3. Change in hardness of unfermented (A – 0% inulin) and fermented (B – 0%, C – 1%, D – 2%, E – 3%, and F - 4% inulin) pea protein ice creams after storage
for 1st, 5th, and 10th day.

5
V.R. Narala, M.A. Jugbarde, I. Orlovs et al.
Table 5
Change in colour of unfermented (A – 0% inulin) and fermented (B – 0%, C – 1%, D – 2%, E – 3%, and
F - 4% inulin) pea protein ice creams after one-day storage.
A B
L∗ 51.01 ± 1.37a 56.59 ± 1.26b
a∗ 4.71 ± 1.33a 9.40 ± 1.5b
b∗ ­3.7 ± 1.28a ­3.20 ± 1.52a
∗
Different superscript letters (a,b,c,d) show significant differences (p<0.05) in a row.
and F added with 2, 3, and 4% of inulin, respectively, had significantly
(p<0.05) lower hardness values than the yoghurt-ice creams B and C (0
and 1% of inulin) in the whole storage time of 1st, 5th, and 10th day.
These results can be attributed to, the added inulin in the formulations
is capable of adsorbing free water molecules during homogenisation and
freezing of ice cream mixes, results in lower ice crystal formation leads
to a softer product. The present study observations are comparable with
the results reported by El-Nagar et al. (2002), who found that the ap-
plication of inulin as a fat replacer in the low-fat ice cream at the level
of 5, 7, and 9% produces a significant reduction in the hardness of ice
cream. The hardness of all the unfermented and fermented pea protein
ice creams in this work was increased with the storage period can be
explained as the formation of larger ice crystal during the storage, the
similar results were produced during the studies Ahmad et al. (2020) and
Guler-Akin et al. (2021).
3.5. Colour
The colour L∗ , a∗ , and b∗ values of the pea protein fermented and
unfermented ice cream samples were measured after 1 day of storage.
The results (Table 5) shows that a lower lightness L∗ value in the unfer-
mented ice cream (A) was obtained due to the colour of blueberry juice
concentration and pea protein isolates. However, the L∗ values were in-
creased after fermentation of the mix and increased with the addition of
inulin. The authors Rankin and Brewer (1998) mentioned that milks fer-
mented with exopolysaccharide producing cultures were scored higher
than non-exopolysaccharide producing cultures with respect to edge and
centre whiteness. Fermentation and exopolysaccharide production may
increase the visual perception of whiteness in milk. The positive redness
a∗ values were observed in all ice creams and it was increased with fer-
mentation. The negative b∗ values of all samples were indicating a blue
colour in the ice creams due to the addition of blueberry juice concen-
tration. Guler-Akin et al. (2021) produced a vegan pea protein-based
ice cream with the L∗ value of around 68 and they mentioned, a∗ and
b∗ values increases with the increase of pea protein isolates due to its
greenish-brown colour and no change in colour of pea protein ice cream
during 60 days of storage period. Sivasankari et al. (2019) found that
the darkness in the pulse protein-based ice cream is due to the Maillard
reaction that occurs during heat treatment.
4. Conclusions
Based on the present research results, inulin can work as a prebiotic
to the culture Streptococcus thermophilus and Lactobacillus bulgaricus in
pea protein yoghurt-ice cream. The addition of inulin to the pea pro-
tein ice cream mix led to a faster drop in pH during incubation due to
its prebiotic activity. Fermentation with yoghurt culture can be useful
for improving the hardness of pea protein yoghurt-ice cream. A higher
overrun of the ice cream can be achieved with the addition of inulin
between 2 and 4% to the mix. The higher viability of yoghurt bacteria
in pea protein yoghurt-ice creams was maintained during 10 days of
storage with 3 and 4% inulin content. Hence, it is possible to develop
a new vegan and functional probiotic yoghurt-ice cream based on pea
protein isolates as an alternative to dairy proteins. But, further investi-
gation on the sensory analysis of the fermented pea protein ice cream
must be done in the view of consumer acceptance.
Applied Food Research 2 (2022) 100136
C D E F
57.93 ± 1.31bc 59.47 ± 1.15cd 60.89 ± 1.78d 61.07 ± 1.18d
9.42 ± 1.24b 9.51 ± 1.43b 9.48 ± 1.25b 9.56 ± 1.13b
­3.31 ± 1.64a ­3.05 ± 1.04b ­2.76 ± 1.43b ­2.81 ± 1.18b
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This research has been conducted by "Nature Foods” Ltd. with finan-
cial support from the European Regional Development Fund (contract
No. 1.2.1.1/18/A/002) within the framework of research project No.4.
- “Development of new and innovative products at Nature Foods” sup-
ported by Latvian Food Industry Competence center. We would like to
thank Dr Ruta Galoburda (Faculty of Food Technology, Latvia University
of Life Sciences and Technologies) for support and providing research
lab facilities.
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