Development of Whey Protein Enriched Banana Based Smoothie

Mohammed Ajlan, Prince Chawla

Department Food Technology and Nutrition, Lovely Professional University, Phagwara, Punjab,
144411, India
2. Materials and method
2.1 Materials
Raw materials
Whey protein concentrate (WPC-80) was obtained from Roquette India Private Limited.
Mumbai, Maharashtra, India. For the formulation of smoothie wholesome Robusta bananas
were collected at their optimum maturity and ripening stage, from local fruits market of
Phagwara, Punjab. Milk was collected from university cattle yard, Lovely Professional
University, Punjab.

Chemicals
All the chemicals such as sodium hydroxide, petroleum ether, potassium sulphate, copper
sulphate, boric acid were obtained from Loba Chemical Pvt Ltd. Mumbai, India. Sulphuric acid,
phenolphthalein and stabilizer as Gum Arabic was obtained from Central Drug House Pvt Ltd.
New Delhi, India. Nutrient Agar was obtained from Hi- media Pvt Ltd. Mumbai, India and
Ethanol was obtained from Changshu Hongsheng Fine 22 Chemical Co. Ltd. Jiangshu (China).
Glassware (Borosilicate) used were class “A” certified Aqua regia washed. Moreover, double
distilled water was used for the preparation of reagents and chemical solutions.

2.2 Methods

2.2.1 Preparation of whey protein enriched banana-based smoothie:

Whey protein enriched banana smoothie was formulated according to the method described by
Srivastava et al., 2019, without adding any artificial sweeteners or emulsifiers or other
chemical preservatives. The preparation was included with 3 steps, i.e., pretreatment,
blending and final pasteurization. In pretreatment steps, the banana was cleaned, peeled and
chopped with a kitchen knife and milk was pasteurized at 72 °C for 15 seconds (Bousbia et
al. , 2021) prior to use. Secondly all ingredients such as whey protein and banana were
weighed according to the ratio of 1:20, 2:30, 3:40, 4:50 (w/w) by maintaining gum arabic (1 g)
and sugar (10 g) as constant. A water-based concentrate of whey protein, gum Arabic and
sugar were made and added into the previously pasteurized milk which was then followed by
blending with the banana by using a juicer for 3-5 min. The prepared smoothie was then
pasteurized to improve the consistency and shelf life. After cooling, the final product was
coded as S1, S2, S3, S4 and kept under refrigeration condition (4- 7 °C) for further analysis.
2.2.2 Sensory evaluation
The sensory evaluation of the smoothie samples was carried out by 15 trained panelists
including students within the premises of the Lovely Professional University, Punjab, using a
nine-point hedonic scale were scores ranged from dislike extremely (1) to like extremely (9)
and water was provided for each panelist for mouth rinsing after each sensation. Thus, the
product was evaluated for its colour and appearances, flavour, consistency, sweetness and
overall acceptability.

2.2.3 Physio-chemical properties

In physio chemical properties, the product was analyzed for proximate analysis according to
AOAC (Association of Official Analytical Chemists) 2010.

Moisture content
The moisture content of product was determined by subjected the sample in hot air oven. 2 g
of sample was taken in a pre-weighed petri dish and kept at a temperature of 50 °C in hot air
oven for 3 h. The loss of moisture content was calculated by weighing the Petri dish with the
sample after every 1 h until constant weight and expressed as % by using the following
equation.

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑃𝑒𝑡𝑟𝑖 𝑑𝑖𝑠ℎ 𝑤𝑖𝑡ℎ 𝑠𝑎𝑚𝑝𝑙𝑒− 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑃𝑒𝑡𝑟𝑖 𝑑𝑖𝑠ℎ 𝑎𝑓𝑡𝑒𝑟 𝑑𝑟𝑦𝑖𝑛𝑔 ×100
moisture % =
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒

Ash content
Ash content in a sample can be determined by weighing 2 g of sample in a pre-weighed silica
crucible and kept on heating mantle until it became smokeless. Then the charred sample was
placed in muffle furnace (Narang Scientific Works Pvt Ltd. New Delhi, India) for a time of 6
h at 550°C. The values were then applied to the below mentioned equation and the % of ash
weight of ash
was determined. Ash %= ×100
weight of sample

Fat content (Soxhlet apparatus)

Fat content was determined with the moisture free sample of 2 g using a Soxhlet apparatus
(Socs Plus, Pelican INC. Chennai, India) in which thimble contains the ). Approximately from
2 g of sample which the fat was extracted using solvent as Petroleum70 ml of petroleum ether
70 ml. The final and the percentage of the fat content was determinedthen calculated by the
below mentioned equation.
weight of fat
Fat %= ×100
weight of sample

Crude fiber
 The crude fiber content was estimated by heating 2 g of defatted sample with 200 ml 1.25 %
H2SO4 for 30 minutes with rotating the flask in every 5 minutes. After that, the mixture was
filtrated through a muslin cloth and resultant residue was washed thrice with double distilled
water. Same procedure was followed with the residue using 1.25 % of NaOH (2.5 g in 200
ml D.D.W) and the final residue was taken in a pre-weighed crucible and oven dried for 3 h
at 50 °C followed by ashing in a muffle furnace at 550 °C for 2 hours. Then the % of fiber
was calculated using the below mentioned equation.

weight of crucible with dry residue × weight of crucible with ash

% of crude fiber = × 100
weight of sample

Protein content
Kjeldahl (Kjeloplus, Pelican INC, Chennai, India) apparatus was used to determine the total
nitrogen content of sample. There were 3 steps in the protein estimation: digestion, distillation,
and titration. Digestion was done using concentrated sulphuric acid and using potassium
sulphate to raise the boiling point. Nitrogen value was calculated along with the protein factor
(6.25) by multiplying with the titrated value. The protein value was then calculated using the
equation below.
normality of acid ×titer value(A−B)
N2 content = 1.4 ×
Amount of sample
N= N2 content × protein factor
where, A was the titratable value of sample and B was the titer value of blank.

Carbohydrate content
Carbohydrates in the sample was estimated by difference method i.e., subtracting the sum of
above- mentioned parameters (moisture, ash, protein, fat, and fiber) from 100 on the dry basis
(moisture free).

pH
The degree of acidity and alkalinity of prepared smoothie were determined by using a digital
pH meter (Labtronics, Haryana, India) (AOAC, 2010) at room temperature (27 °C). Before
analysis pH meter was calibrated using various buffer solution (pH 4,7,9) then followed by the
diluted sample with double distilled water (1:1 v/v).

Titratable acidity
The titratable acidity of the sample was determined by using AOAC, 2010. About 1 ml of
formulated smoothie was dispersed in 9 ml of double distilled water and titrated against 0.1 N
NaOH by using Phenolphthalein as an indicator. From the obtained titratable value, the acidity
of the sample was calculated by using the following equation.
 normality × titratable value
Titratable acidity= ×100.
volume × specific gravity

Energy value
The energy value of the sample was determined using Bomb Calorimeter (Labtronics,
Haryana, India) according to Roberts et al (2018) to see the calorific value. The unit mass of
sample was burnt, and the heat evolved was allowed to absorb in water, where it was recorded
as function of temperature. The energy of the sample was estimated by adding 1ml of sample
into the crucible and hence it was found.

Viscosity
According to Markowski et al (2017) to determine the viscosity of the sample Brookfield
viscometer (Labtronics, Haryana, India) was used. The spindle used in the viscometer was LV
spindle set with spindle number S64 attached to the viscometer by screwing them with the
lower shaft which was levelled on the platform. The viscometer was operated at different RPM
(6, 12, 30, and 60) and hence the values were noted. The value of viscosity was then calculated
using the equation below.
tsρs
ηs = ηw ×
twρs
where,
ηs = viscosity of the solution
ηw = viscosity of water
ρs and ρw = density of solution and water respectively
ts and tw = time of solution of water.

2.2.4 Characterization of product

Fourier Transform infrared (FTIR) spectroscopy

WHAT IS FTIR ?

WHY WE USE FTIR?

According to Li et al (2020) functional groups of bioactive components in smoothie were

evaluated by spectrophotometer (PerkinElmer, Massachusetts, USA). Multiple scans emulsion
sample were achieved against air in the mid-infrared region 4000-400 cm-1. Thus, the
transmittance was obtained in Data by using the software PerkinElmer SpectrumTM 10.
Differential Scanning Calorimetry (DSC)
The thermal property of formulated protein-phenol complex of smoothie was determined by
Differential Scanning Calorimeter (PerkinElmer, Massachusetts, USA) according to Bhambar
et al (2021) analyzed in powdered form with thermocouple-based temperature sensors and
with a nickel-chromium sample plate Heat from -30.00°C to 200.00°C at 10.00°C/min.
2.2.5 Microbial stability
Total Plate Count
Total Plate Count was determined by dissolving 1 g of sample in 9 ml double distilled water
for performing serial dilution (Dilrukshi et al., 2021). Nutrient agar of 2.8 g was added to
the conical flask by adding 100 ml double distilled water. Autoclave the petridish, test tubes
and conical flask at temperature of 121 °C with 15 Psi for 15 min which was covered with
brown sheet.
2.2.6 Statistical Analysis
In this study each experiment was performed in triplicate and results were reported in mean
and standard deviation by analyzing data using ANOVA.