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 American Journal of  
Food Science and Technology (AJFST)

Development of  Functional Nutritional Whey Beverage with Herbal Extract and Its 
Sensory Quality

Ambitama Guha1, Aditi Roy Chowdhury1*

Volume 3 Issue 2, Year 2024
ISSN: 2834-0086 (Online)

DOI: https://doi.org/10.54536/ajfst.v3i2.2855
https://journals.e-palli.com/home/index.php/ajfst

Article Information ABSTRACT

Received: May 02, 2024

Accepted: June 08, 2024

Published: June 12, 2024

The dairy industry is one of  the biggest Food Industries worldwide. In the dairy industry, lots 
of  whey is during the Paneer manufacturing process. In local sweetmeat production units 
of  India, a huge amount of  whey is not handled for preservation and is discarded locally 
which increases the BOD level of  wastewater. The objective of  the current work is to make 
a value-added product utilizing dairy waste with the inclusion of  natural preservatives in the 
form of  herbal extracts. Transforming the whey into a value-added product is addressed in 
the present study. The management of  such waste may generate a beneficial product (Herbal 
whey beverage) with numerous health benefits and it may be recognized as a functional whey 
beverage worldwide. The methodology of  the production of  whey was followed using the 
natural curdling agent in hot conditions with cow milk and cooled down immediately. Herbal 
extracts were prepared and mixed with whey, maintaining a proper ratio, and different trial 
runs were done. The final pasteurization step was carried out with a specific time-temperature 
profile. The final finished product was hot-filled in a 200 ml sterile glass bottle and corked. 
The production, preservation, and overall shelf-life study were undergone in the current 
work.  The preservation concept was addressed with some natural preservatives using herbs 
like Cumin, Cinnamon, and Mint for their antimicrobial properties. The observation of  the 
current work satisfactorily claims that the developed beverage is microbially safe for up to 4 
weeks when kept in a refrigerated condition. The sensory analysis and chemical analysis also 
suggest the product’s goodness up to 4 weeks with all attributes of  sensory parameters like 
haze, pH, brix, colour, flavour, taste, and mouth-feel. All the operational steps were handled 
hygienically.

Keywords

Herbal Beverage, Whey, 
Preservation, Cinnamon, 
Cumin, Mint, Sensory Analysis

1 Department of  Food Technology, Techno Main Salt Lake, EM 4/1, Sector-V, Kol-91, India
* Corresponding author’s e-mail: rcrakhi29@gmail.com

INTRODUCTION
The dairy industry is considered to be one of  the 
largest industries in the food sector all over the world. 
Lots of  valuable products from this sector starting 
from pasteurized packaged milk, milk powder, ready-
to-eat milkshakes, cheese, paneer, butter, ice cream, 
etc are currently available in the world market. Among 
these products from milk, cheese has been the largest 
produced milk-based product that creates a by-product, 
whey. Whey is the largest produced by-product from milk 
processing or cheese processing, which is the liquid part 
that separates after the milk proteins’ coagulation. In the 
Global statistical result, it has been seen that in 2022 cheese 
production has amounted to about 22.17 million metric 
tons. Among these, the European Union produces about 
10.55 million metric tons of  cheese per year claiming 
itself  as the top cheese producer (Statista, 2023, Cheese 
production worldwide 2015-2023). Out of  the total 
production of  liquid whey only 3.2 million metric tons 
have been utilized by industries to develop some value-
added products, whey protein concentrates, isolates, etc. 
In recent years whey-based beverage production includes 
whey proteins which have been introduced even in the 
US market (Chavan et al., 2015), (Miller, 2009).
The expectation for the Global production of  cheese 
has reached 168 million tons of  cheese whey and 50% 
of  that whey gets wasted as animal feed, biofertilizer 

in irrigation systems, and discharged without treatment 
(Pires et al., 2021). But despite being a by-product whey 
has been proven to have immense health benefits. Whey 
can be considered a liquid enriched with nutrients. The 
composition of  whey is equivalent to 94% of  water and 
the total solids remain at 6%. Whey contains lactose (4.5% 
of  total whey), 0.8% proteins, and 0.7% minerals (Pires et 
al., 2021). Even though whey proteins are present in small 
amounts they show a high PER value (Protein Efficiency 
Ratio) and, a high BV value (biological value). The Net 
Protein utilization score is almost 95 for whey proteins 
which are the source of  α-lactalbumin, β-lactoglobulin, 
casein-peptides, lactoferrin, immunoglobulin, and 
lysozyme. These varied compositions in whey proteins 
give overall immunity, anticancer effects, and show 
resistance to pathogenic activity. The whey proteins 
have also recorded antimicrobial properties (Gottschalk, 
2005). The other valuable components are mentioned by 
(Tsermoula et al.,2021) as nitrogenous matter like whey 
protein, non-protein nitrogen, and peptides. The proteins 
present in whey β-Lactoglobulin and α-lactalbumin are 
known as immunity proteins. Minor proteins such as 
lactoferrin which is an iron-binding glycoprotein and 
found to be present at a 3% level of  total whey protein. The 
functional components present in whey are in the form 
of  oligosaccharides with a minor amount of  glucose and 
galacto-oligosaccharide (Hernández-Ledesma et al.,2011). 



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Some functional micronutrients are also present in whey 
like minerals, mainly calcium, phosphorus, potassium, 
sodium, chloride, and magnesium. Some vitamins are also 
found to be present in whey such as Complex B vitamins, 
especially Riboflavin B2 and Vitamins B12. Polar lipids 
are also found to be present in whey with an admissible 
amount (Tsermoula et al., 2021).
When whey has been discarded from industries as run-
off  effluent (without any treatment), it will impart water 
pollution like an increase in BOD level, degrading the 
water quality index. Not only BOD the increased amount 
of  discarded whey may also give rise to the COD level 
as high as 50gms to 80gms COD L-1 or 40gms to 60gms 
BOD L-l.  Impact of  a small creamery or sweet meat shop 
can produce a high amount of  BOD value per day. which 
requires a huge amount of  expenditure for treatment, 
mostly whey is discarded untreated. (de Almeida et al., 
2021).
As whey has been proven to be a nutritious liquid, 
researchers have recognized whey as a co-product rather 
than a by-product of  the cheese, and paneer industries. 
Whey-based products that are found in the recent market 
as whey beverages include fermented and non-fermented 
fruit beverages, alcoholic whey beverages, sports drinks, 
whey protein powders, and isolates (Papadem & Kotsaki, 
2019).
The current study has been focused on developing whey-
based beverage with the inclusion of  some indigenous 
herbal extracts. Mint, Cinnamon, and Cumin were 
selected to prepare the herbal extractive to preserve the 
whey on the basis of  the concept of  natural preservation 
method without the addition of  any preservative. 
These indigenous herbs contain phytochemicals like 
polyphenols, flavonoids, tannins, alkaloids, terpenoids, 
lectins, etc. (Viji et al.,2015).
Cinnamon has a definite role in lowering blood glucose 
levels and can modulate insulin levels. It is associated with 
the reduction of  type 2 diabetes. Cinnamaldehyde which 
is the active component in cinnamon has antimicrobial, 
antioxidative, and anti-inflammatory effects. Cell 
structure studies provide proof  that it inhibits the growth 
of  bacteria associated with food contaminants (Singletary, 
2008).
The antibacterial activity of  cumin is well recognized for 
its active components; alkaloids, flavonoids, glycosides, 
saponins, and organic acids (citric acid, malic acid, 
tartaric acid, ascorbic acid, propionic acid) (Clark, 1998). 
Cumin (cumin aldehyde) is proven to be highly packed 
with antioxidants and antimicrobial properties that work 
against both gram-positive and gram-negative bacteria 
along with some fungi groups (Kedia et al., 2014), (Belal  
et al., 2017).
Considering the valued nutritious components, functional 
oligosaccharides, immunity proteins, essential minerals, 
and vitamins, the whey-based beverage was aimed in 
this research to serve as a valorized product for the 
community in the tropical and subtropical zone. In South 
Asian countries including India, there is a huge market for 

herbal beverages (Dini, 2019).
The preservation of  whey with the natural mode was 
focused in the present study in a blend of  herbal extracts 
like cinnamon, cumin, and mint. The availability of  herbs 
is common in Indian scenarios and it is also cost-effective. 
The current study investigates the extended shelf-life 
study of  whey with herbs having their bacteriostatic 
action with a view to less heat treatment for a quality 
product.
This research aims towards a green technology and 
sustainable approach towards the exploitation of  whey. 
The final product is decided as a ready-to-drink beverage 
with a new preservation mode/concept because of  the 
high demand for beverages in hot and humid climates. 
This beverage could be also considered as a valorized 
product in the post-Corona episode or can drive a parallel 
beverage sector towards a nutritional aspect compared 
with carbonated drinks.

MATERIAL AND METHODS
Materials
Standardized and Pasteurized cow milk of  a recognized 
Indian brand with 4.5% fat and 8.5% SNF was purchased 
for whey extraction, from a local market in Kolkata. 
Sugar, lemon, and salt were also purchased from a local 
market in Kolkata, India as well. The choice of  Herbs 
were fresh mint leaves, whole cinnamon, and whole jeera 
of  a local brand in Kolkata, which were also purchased 
for the preparation of  the trials, from a local grocery 
shop in Kolkata, India.

Preparation of  Whey
The whey has been extracted using the traditional acid 
curdling method, since the ultimate objective is to use 
all the natural ingredients rather than citric acid, lemon 
juice has been used as the natural source of  citric acid 
for the coagulation of  casein protein. The processing of  
whey was done by taking 1L of  milk, heated up to 80° 
C followed by the addition of  lemon juice for curdling 
operation. The process was continued for near about 
30seconds for the whey to be separated.
After the separation of  the whey from the casein protein 
the separated whey was collected in a sterile beaker using 
a muslin cloth and then sealed with aluminium foil, and 
refrigerated. The pH of  the raw whey was taken at 25°C 
with pH paper (Merck Life Science Pvt. Ltd) which came 
out to be 6 and the brix was measured with a refractometer 
which came out to be 7°.

Preparation of  the Extractives
Three extractives have been prepared that are extractive 
of  mint, cinnamon, and cumin. For the extractive 
preparation coarsely grounded cumin and cinnamon in a 
grinder and freshly mashed mint leaves in a mortar pestle, 
were taken. Each extractive was taken in three separate 
muslin clothes. In three different beakers, 250ml of  water 
was been heated in a double boiler method up to 60°C. 
Each of  the herbs, packed in muslin cloths, was then 



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dipped in three separate beakers for 5 minutes maintaining 
the temperature at 60°C. After the preparation of  three 
extractives, the beakers with the extractives were sealed 
with aluminium foils and then refrigerated.

Preparation of  the Final Beverage
The final beverage was prepared by mixing the whey and 
the herbal extract in a proper ratio. Salt and sugar have 
been admixed according to taste. The beverage has been 
pasteurized for 1 minute at 90°C. After pasteurization, 
the beverage was filled in a sterile glass bottle of  200ml 
and then refrigerated at 10±2°C. 

Formulation of  Trials
Two trials have been done by taking different ratios of  
whey and herbal extractives. In Trial-1 the proportions of  
the ingredients were 70ml whey and 30ml of  extractives 
with salt and sugar. In Trial-2 the proportions of  the 
ingredients were 60ml of  whey with 40ml of  extractives 
with salt and sugar. Two trials were made based on varying 
the whey along with the herbal extract proportions on 
a preliminary basis and the final formulation has been 
selected based on the sensory scores. The shelf-life study 
was done on the final product.

Sensory Analysis
The final formulation of  the beverage has been finalized 
using the hedonic scale based test of  each trial. The test 
was carried out among four different age groups: (less 
than 20), (20- 40), (40-60), and (above 60) making a three-
member panel for each age group, the trials were tasted 
with a triangle test (Chude et al., 2023).

Chemical Analysis
Proximate analysis was carried out following (A.O.A.C., 
2010). Instruments utilized in the Chemistry of  Food 
laboratory, TMSL named Hot air oven (Instrumention 
India), muffle furnace (Instrumention India), Water 
bath, Heating mantle, pH Meter (Toledo), and Weighing 
machine (Mettler), and Spectrophotometer (Systronics).

Shelf-Life Study
A shelf-life study was done on the sample rated high by 
the panellist in sensory analysis. The shelf-life study was 
undergone by keeping the corked bottle in refrigerated 
condition at around 10±2°C for 4 weeks. The pH, brix 
flavour, haze, colour, appearance, consistency, and also 
the chemical analysis such as total sugar, total solids, 
titrable acidity (in terms of  lactic acid), antioxidant (in 
terms of  gallic acid equivalent), and soluble protein 
content were investigated between intervals of  7 days, of  
the total of  the 4 week study was to get an idea for the 
shelf  life of  the prepared processed product (Khasanov 
& Matveeva, 2020).

Microbial Analysis
Microbial analysis was done by pour plate method 
with Nutrient Agar media for the bacterial count. The 

plates were kept in the incubator at 42°C for 48 hours 
maintaining the time temperature ratios for colonisation. 
Colonies were counted using the colony counter. By 
using Czapek Dox Media yeast and mould counts were 
taken. The plates were kept in the incubator at 25°C to 
30° C for 48 hours maintaining the time temperature 
ratios for colonization and the colonies were counted 
using the colony counter. Overall work is done taking all 
the necessary precautions in the microbiology laboratory 
of  TMSL, MOFPI (Chowdhuryet al.,2023).

Antioxidant Analysis
1ml (1:20) diluted beverage sample has been taken in 
a conical flask followed by the addition of  1ml follin-
ciocalteu reagent (1:5 with water) and kept aside for 
5 minutes. Addition of  3ml 20% Na2CO3 was done 
and the sample was kept for 30 minutes in a dark 
environment and absorbance was recorded at 760nm 
in a Spectrophotometer (Systronics). The water-soluble 
antioxidants (bioactive components) were analyzed in 
terms of  the gallic acid equivalent of  the final beverage 
which is Trial-2 (Chowdhury et al., 2023).

Thin Layer Chromatography (TLC) for Qualitative 
Analysis of  Essential Amino Acids
Thin Layer Chromatography has been done using TLC 
Silica Gel 60 F254 25 Aluminium Sheets (Merck KGaA) 
and solution preparation, (Butanol: Glacial Acetic Acid: 
Water in the ratio of  4: 1: 1) has been taken. The known 
essential amino acids along with the beverage sample 
were been spotted on the aluminium TLC sheets and 
were kept aside to let the solution flow along with the 
essential amino acids and the beverage sample. Ninhydrin 
is used to spot the flow or the distance travelled by the 
essential amino acids (Bele & Khale, 2011).

RESULT AND DISCUSSION
Table 1 comprises the whey volume, along with the 
curdling temperature, brix, and pH; which are to be 
recorded for the generation of  the final finish product 

Table 1: Processing of  whey
Total whey extracted from 1L of  
milk

700ml (Approx)

Curdling Temperature 80°C
Brix 7.1
pH 6

which may be the contributed fraction with the herbal 
extractives.
(All data shown in Table 1 and in all other Tables are the 
mean of  the triplicate values).
Table 2 gives a view of  the extractive preparation with 
the herbs (cinnamon, cumin, mint). Figure 1 shows 
the extractives that have been prepared in the Food 
Processing Laboratory of  TMSL. The preparation of  
the extractives with the heterogeneous herbal fractions 
was observed with the values, almost the same for all 



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fractions with respect to the temperature and time profile, 
kept constant for every extractive preparation step along 
with the curdled whey which has been developed in the 
laboratory of  TMSL processing lab. Hygienic practices 
have been employed to handle the perishable items with a 
keen monitoring for production.

report has been given with the strawberry-based drink 
where the pH was recorded as 6.5 to 6.7 with sugar and 
additional flavour notes, whereas for fermented products, 
sour milk, buttermilk, and kefir the pH was adjusted to an 
acidic range of  4.8 to 4.5 (Athanasiadis, I., et al., 2004).
The pH value of  the product of  the current study has 

Table 2: Processing of  whey
Cumin Extractive      

Composition
Cinnamon Extractive      

Composition
Mint Extractive      

Composition
Water 250ml Water 250ml Water 250ml
Herb 10gm Herb 10gm Herb 10gm
Time 5minutes Time 5minutes Time 5minutes
Temperature 60°C Temperature 60°C Temperature 60°C
Brix 1° Brix 1° Brix 1°

Figure 1: Extractives (Mint, Cumin, Cinnamon)

Table no. 3 contains the pasteurization time and 
temperature of  500ml final beverage which is 1 minute 
at 90°C. The beverage has been manufactured following 
Good Manufacturing Practices. Since all the protocols 
have been maintained the time for pasteurization has 
been kept low. The low time for pasteurization is also a 
measure taken to preserve the high-valued whey protein 
at its best condition. (Vajdi & Pereira, 1973) also reported 
almost the same time temperature profile with strawberry, 
lemon, chocolate, inserted whey drinks, the overall time 
temperature condition for the mixture was maintained at 
82°C for 2 minutes, which is almost similar to our study.
Table no. 4 consists of  the total bottling amount which 
is 200ml. The brix has been maintained to be 15° as per 
FSSAI beverage guidelines (Food Products Standards and 
Food Additives Regulations, 2011). The pH has been kept 
at 5.4 which inhibits microbial growth that is the parameter 
pH has been used as a hurdle for microbial growth. A 

Table 3: Pasteurization time temperature of  Final 
Beverage
Pasteurization Amount 500ml

Time 1minute
Temperature 90℃

Table 4: Soluble solid vs pH of  the Final Beverage
Bottling amount 200ml

Brix at 30℃ 15°

pH at 30℃ 5.4

Table 5: Trial 1- Formulation with 70% whey and 30% 
Herbal Extractives
Ingredients Fractions
Whey 70ml
Cumin extract 15ml
Cinnamon extract 10ml
Mint extract 5ml
Salt 40gm
Sugar 90gm

Table 6: Trial 2- Formulation with 60% whey and 40% 
Herbal Extractives
Ingredients Fractions
Whey 60ml
Cumin extract 25ml
Cinnamon extract 10ml
Mint extract 5ml
Salt 40gm
Sugar 90gm

Figure 2: Final Beverage, (Whey with Herbs)

been recorded as 5.4 which may be corroborated with the 
previous report.
The combination of  whey and extractives were continued 
with two trials which are reflected in Table 5 (Trails-1) 



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and Table no. 6 (Trial-2). The objective of  the trial runs 
were to depict the idea about the acceptability of  the 
finished product with respect to the sensory score. The 
sensory evaluation is done with respect to taste, flavour, 

mouthfeel, and appearance. These data reports are given 
in Tables No. 7 and 8.
The brix value of  the final finished product was estimated 
at 15° brix (FSSAI Regulations, 2011) for preservation 

Table 7: Hedonic Rating for Trial-1
Age Group Taste Flavour Appearance Colour Mouthfeel Overall Impact

6.5 7 6 5.5 5 5.8
7 7 5.5 5 5
7 6.5 5 5 5

Mean 6.8 6.8 5.5 5.2 5.0
20-40 6.5 6 6 6 6 6.2

6 7 5.5 6 6
6 7.5 5.5 6 6

Mean 6.2 6.8 5.6 6.0 6.0
40-60 7 7 6 6 6 5.9

6 6 6 5.5 5
6.5 5 5.5 6 6

Mean 6.5 5.9 5.8 5.8 5.6
Above 60 5 5 5 6 6 5.7

6.5 5.5 6 6 6
7 6.5 5.5 5.5 6.5

Mean 6.1 5.6 5.5 5.8 6.2

Table 8: Hedonic Rating for Trial-1
Age Group Taste Flavour Appearance Colour Mouthfeel Overall Impact
Less than 20 7 6.5 6 7 6.5 6.6

8 8 6.5 7 6
7 7 5.5 6.5 6

Mean 7.3 7.1 6 6.8 6.2
20-40 7 7.5 6 6.5 7 6.7

8 8 6.5 5 7
7.5 7.5 5.5 5.6 6.5

Mean 7.5 7.6 6 5.8 6.8
40-60 5 6 5 6.5 7 6.4

7 7.5 5.5 6 8
6.5 7 6 7 7

Mean 6.1 6.8 5.5 6.5 7.3
Above 60 6.5 7 5.5 7.5 7 6.6

8 6.5 5 6 7.5
7 6.5 5 7 7

Mean 7.1 6.6 5.1 6.8 7.2

purposes and also for taste. The pasteurization time and 
temperature were adjusted for the finished product to 1 
minute at 90℃ that data can be corroborated with (Vajdi 
& Pereira, 1973). The holding time for pasteurization 
temperature was 1 minute in a closed vessel to avoid 
the oxidative changes of  the smaller peptides and also 
to minimize the loss of  the volatiles from the herbal 
fractions. Further, the time-temperature profile might 
be affecting the composition of  the finished product; 

the derivatized product of  the sugar base, until we are 
not controlling the exposure to high pasteurization 
temperature for a longer period.
Figure 3 shows the sensory score data for both trials. The 
result shows that Trial 2 (60:40 ratios of  whey: herbal 
extracts) had higher sensory scores among the people 
category of  the age group of  less than 20, 20-40,40-
60, and above 60 years. The sensory score was pointed 
towards the high scale value with the sensory parameters 



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shown with respect to taste first for all categories. Whereas 
the colour effect for every group went down to a limit 
value of  5.8. When the sensory scores are evaluated the 
overall appearance of  the product is almost the same. 
The overall acceptability score for Trial-2 was higher than 
Trial 1 shown in Table 7 and 8. 
Figure 4 represents the most liked parameter in the final 
trial which is Trial 2. Among the panelists from the age 
group less than 20, taste is the most accepted parameter. 
Among the panelists from the age group 20-40, smell is 

the most accepted parameter whereas among the panelists 
from the age group 40-60 and above 60, mouthfeel is the 
most accepted parameter. The compositional variation of  
Trial 2 was higher with plant-based extract in comparison 
with Trial 1. The animal-based by-product (whey) has 
been mixed with plant-based extracts and was controlled 
to develop a designer food (whey-based herbal beverage) 
the final product’s storage and its shelf  life were judged.
The chemical analysis report of  the developed product 
was preserved for a longer period in refrigerated condition 

Figure 3: Best scores of  the parameters from the comparison of  each trial

Figure 4: The most liked parameter from the final trial that is Trial-2

Table 9: Chemical Analysis for shelf-life study
Chemical Analysis 0th day Week 1 Week 2 Week 3 Week 4
Temperature (±2) 10 ºC 10 ºC 11 ºC 12 ºC 12 ºC
pH 5.4 5.4 5.4 5.4 5.5
Brix 15° 15° 15° 15° 15°
Total Sugar (After Inversion) 13.66% 13.66% 13.66% 12.9% 12.86%
Total Solid 15.1% 15.1% 15.2% 14.8% 14.8%
Titratable Acidity (in terms of  lactic acid) 0.21% 0.21% 0.20% 0.21% 0.22%
Ash 0.26% 0.26% 0.25% 0.25% 0.24%
Soluble Protein mg/ml 6.75  6.75  6.70   6.65   6.66



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kept at 10±2℃ in a corked glass bottle is depicted in 
Table 6. Upon storage for 4 weeks there were not any 
significant changes in pH value (5.4,5.4,5.4,5.5) starting 
from the 0th day, 7th day, 15th day, 21st day, and 30th day, 
and the recorded pH are given in Table 9, normal pH 5.4-
5.5. This data can be corroborated with the data given by 
(Binsi et al, 2007), where we found no significant changes 
in pH with a marginal value reaching 6.58, 6.59, and 6.61 
with the plant extract treatment starting from the initial 
day. As there is no increased value in pH observed upon 
storage, it expresses that loss of  volatiles is being arrested 
with the effect of  the plant extract which can control the 
pH values for up to 15 days on chill storage (Gao et al., 
2014). The investigated results showed that the present 
study depicts that pH is not a good indicator of  spoilage 
for the early 15 days.
Table 9 suggests that the total solid content does not have 
any significant change within the established acceptable 
period starting from 0 days to the 30th day. The recorded 
soluble solid value was observed through a refractometer 
as a 15° brix (Brix has been considered as the soluble 
solid content). The result of  the current study may 
suggest that the soluble solids (soluble proteins, soluble 
sugar, soluble metal ion, soluble plant extracts) do not 
show any effect upon the beverage consistency and also 
show some controlling effect on the bacterial growth 
during the storage in chilled condition. The total sugar 
was measured after doing inversion of  the preserved 
sample and recorded weekly the total sugar in terms 
of  glucose equivalent was recorded as 13.66%, 13.66%, 
13.66%, 12.9%, 12.86% based on the 0th day, 7th day, 15th 
day, 21st day, and 30th day.
The developed product of  the present work consists of  
different herbs and their extracts along with a certain 
volume of  whey. Cumin contains essential oils that are 
well-recognized for antimicrobial effects, especially 
against S. aureus and E. coli (Monteiro-Neto et al.,2020). 
The extractives prepared from coarsely ground cumin 
powder have shown an inhibitory effect on microbial 
growth as said in (Škrinjar & Nemet, 2009), in the 
developed product, where cumin extract was the primary 

component in the herbal extracts that are admixed to 
develop the product (a whey-based herbal drink). The 
naturally occurring antimicrobial component present in all 
three extractives used in this current work that is Cumin, 
Cinnamon (Asimi et al,.2013), (Diniz do Nascimento et al., 
2020), and mint (Soleimani et al., 2022) might have some 
controlling factor for giving a better shelf  life for the 
developed product this can be exemplified by the use of  
natural bioactive components towards the conventional 
preservation mode of  operation.
The recorded acidity was maintained for a 1-month tenure 
from 0th day as (0.21%, 0.21%, 0.20%, 0.21%, 0.22%) and 
the titrable acidity was measured in terms of  lactic acid 
equivalent. The acidity will be the index for maintaining 
a well-preserved product. The current beverage which 
consists of  whey along with herbal extracts may be a 
ready food source of  microorganisms. The hurdles like 
acidity have to be given properly so that bacteria, fungi, or 
protozoa can be inhibited. The observed data for acidity 
for a month shows that it is more or less constant. And 
thereby preventing spoilage and maintaining the quality 
of  the preserved product.
Mineral content was measured in terms of  total ash of  
the developed products and recorded from the 0th day 
to 30th day as (0.26%, 0.26%, 0.25%, 0.25%, 0.24%) 
this indicates the product is a ready source of  minerals 
as it is the ready source of  natural extractives such as 
the phytochemicals which are coming from cinnamon 
(Khalisyaseen & Mohammed, 2021), cumin (Yessuf, 
2015), and mint(Mimica-Dukic & Bozin, 2008) are mostly 
flavonoids, glycosides saponins, tannin vit. B complex vit. 
E Vit. A Vit. C, alkaloids, carotenes, zeaxanthin, resins. 
Ash content is quite good in cumin, mint, and also in 
cinnamon, which has been reflected in the total ash 
content of  the developed product.
Soluble protein content was measured with respect to 
bovine serum assay which was set as a standard in drawing 
the calibration curve. The known concentrations were set 
as 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml, and 1mg/
ml on the x-axis, and the corresponding absorbance were 
plotted against the y-axis which is depicted in figure 5. The 

Figure 5: Calibration graph for soluble protein with BSA



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Am. J. Food. Sci. Technol. 3(2) 18-29, 2024

concentration of  the soluble protein of  the developed 
product was recorded as 6.75mg/ml value. The process 
whey-based beverage was filled in a 200ml capacity glass 
bottle where the measured soluble protein content was 
recorded as 1.35gm. This protein indicates the combination 
of  soluble proteins which are identified as immunity 
proteins. Hence the developed product may be labelled as a 
rich source of  functional proteins (Solak, B. B., & Akin, N. 
2012). During the shelf-life study of  up to 4 weeks, there is 
no significant changes occurred in soluble protein content.
Shelf-life study of  the developed product has been 

illustrated in Table 10. The preserved product was kept in 
a corked glass bottle of  200ml capacity at a temperature of  
10℃ ±2 for a period of  one month tenure. Smell, colour, 
taste clarity or haze, and consistency were measured 
with very little change. All the parameters except haze 
doesn’t have any additional impact on the final product 
quality. In the case of  haze up to 4th-week data, a small 
amount of  haze was distinguishably observed throughout 
the 1-month period. The antioxidant content, pH value, 
titrable acidity, colour, smell, and taste, these were almost 
comparable with the changes found by the co-workers 

Table 10: Shelf-Life Study
Week Smell Colour Taste Haze Consistency
1st No of  flavour No changes No taste change Little haze at the bottom of  the 

bottle
Homogenous

2nd No of  flavour No changes No taste change Little haze at the bottom of  the 
bottle

No separation of  
water from the phase

3rd No of  flavour No changes No taste change Little haze at the bottom of  the 
bottle

No separation of  
water from the phase

4th No of  flavour No changes No taste change Little haze at the bottom of  the 
bottle

Consistency was as 
good as the first week

in the field of  (Khasanov & Matveeva, 2020). Up to 30 
days value there is a minimum decrease in the value of  
antioxidant content but when stored at a low degree 
temperature which is very similar to our observed result 
with respect to colour taste and flavour and antioxidant 
content when stored at cold temperature conditions.
Microbial analysis has been recorded in Table 11. The 
bacterial plate count was measured in two different 
serial dilutions,10-1 and 10-3 which gives a clear concept 

of  mostly hygienic practices with the recorded count in 
a colony counter. The highest count up to the 4th week 
was recorded as 10 and 3 for 10-1 and 10-3 respectively. 
Whereas the plate counts for yeast and mould showed up 
to the 4th week were 2 and 3 for 10-2 and 10-4 respectively 
counted in a colony counter. Plate count for 10-1 bacterial 
plate count and 10-4 yeast and mould plate count are 
shown in Figures 6 and 7.  As the cold preservation has 
been maintained for 4 weeks duration the growth of  the 

Table 11: Microbial Analysis
Weeks Bacterial plate count 

at 10-1 dilution
Bacterial plate count 
at 10-3 dilution

Yeast and Mould plate 
count at 10-2 dilution

Yeast and Mould plate 
count at 10-4 dilution

Week 1 2 1 0 0
Week 2 3 1 0 0
Week 3 3 2 1 1
Week 4 10 3 2 3

organisms is arrested. The data can be corroborated with 
(Ismail, 2011).

Antioxidant Analysis
The report of  spectrophotometric analysis of  total 
polyphenolic content, in the developed product was 
measured with respect to the gallic acid equivalent is given 
in the given in Table 7. The standard calibration curve of  
gallic acid has been illustrated in Figure 8. On the 0th day 
of  the production, the polyphenol content is recorded in 
the whey beverage as 3.25mg/ml (0.325gm per 100ml) 
which reflects a quite high value of  polyphenol content 
of  the developed beverage. As the preservation study 
was carried out in the current research, we find a less Figure 6: Bacterial plate count of  10-1 dilution                       



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Am. J. Food. Sci. Technol. 3(2) 18-29, 2024

Figure 7: Yeast and Mould plate count for 10-4 dilution

Figure 8: Calibration curve for Total Phenolic Content

depletion of  antioxidant value up to 4th week of  storage. 
This indicates that chilled storage might have some 
effect on the antioxidant capacity value. The developed 
beverage can be said as a healthy beverage keeping a good 
amount of  soluble pigments, total polyphenolics.
The TLC value has been carried out with respect to the 
essential amino acid along with glutamic acid and aspartic 
acid with the prepared standard which are mentioned in 

Table 12. The image of  the TLC sheets with the amino 
acids are given in Figures 9,10,11 and 12. Comparing 
the retardation factor of  all amino acids with the sample 
analyte (whey-based beverage), the predominant amino 
acids which are found to be present are recorded as leucine, 
lysine, threonine, glutamic acid, aspartic acid, and proline. 
The thin layer chromatography method could assess the 
nutrition aspects of  the whey-based beverage in relation 

Table 12: Total Polyphenolic/Antioxidant Content in the Final Beverage
Weeks/Days Polyphenolic Content (In Terms of  Gallic Acid 

Equivalent) mg/ml
0th day 3.375
Week-1 3.375
Week-2 3.375
Week-3 3.12
Week-4 3.06

Table 13: TLC Report
Amino Acid Solute Front/Solvent Front (mm) Experimental Retardation 

Factor (Rf)
Rf  of  beverage (u)

Histidine (A) 6/62 0.096 0.24
Isoleucine (B) 39/62 0.62



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Leucine (C) 20/62 0.32
Lysine (D) 18/64 0.28
Methionine (E) 35/64 0.54 0.23
Threonine (G) 20/64 0.31
Phenylalanine (P) 34/64 0.53
Tryptophan (H) 42/66 0.63 0.32
Valine (I) 34/66 0.51
Glutamic Acid (G1) 20/62 0.32 0.22
Aspartic Acid (X) 13/62 0.20
Proline (Y) 20/62 0.32

to functional health drinks and some sensory attributes 
with respect to the presence of  varied essential amino acid 
content. The qualitative test method has been adopted 

to find the nutritional aspect of  the developed health 
beverage the result in concurrence with (Jain et al., 2013)

Figure 9: Amino acids A, B, C, D, and beverage sample 
U

Figure 11: Amino acids H, I, and Beverage Sample U 

Figure 10: Amino acids E, P, G beverage sample U                                                                                              
and Beverage Sample U

Figure 12: Amino acids G1, X, Y Beverage Sample U

CONCLUSION
Ready-to-serve beverages with the inclusion of  
herbs in dairy wastes (whey) can be a ready solution 
with some health benefits that have been identified 
through current research. The shelf-life study of  the 

said product was observed for 4 weeks without major 
alteration of  food concerning sensory, chemical, and 
microbial qualities. The developed beverage may be an 
alternative sustainable product without the addition of  
any chemical preservative, rather than preservation done 



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with the concept of  bacteriostatic action of  herbs like 
Cumin, Mint, and Cinnamon. In the conclusive part, the 
investigated result suggests that the presence of  a good 
amount of  polyphenols along with a high amount of  
soluble proteins may suggest the developed beverage 
could be mentioned as a functional beverage as per the 
chemical analysis. The developed beverage may also be 
utilized as a ready source for the supply of  carbohydrates, 
soluble proteins, electrolytes, and antioxidants. For 
rehydration purposes, this beverage can effectively cater 
to all age groups including sports persons and elderly 
people for better fluid retention. Not only that that, 
developed beverages will satisfy some of  the essential 
amino acids score within the human system and may 
be commercialized to formulate sports drinks. The 
best preservation method for storage and supply chain 
operations of  the developed beverage is to be maintained 
at a low-temperature condition. Good hygienic practices 
are maintained to develop a value-added product with 
the herbal extracts during processing operations so as to 
create a temperature and time profile in the method of  
pasteurization. A hurdle technology has also been applied 
by maintaining proper acidity, brix value, and inclusion of  
herbal extractives for developing a sustainable Functional 
Nutritional Product (FNP).

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