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Physico-chemical, sensory and microbial characteristics of fruit flavoured milk

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 Adv. Biomed. Pharma. 2:1 (2015) 32-39

Advances in Biomedicine and Pharmacy

(An International Journal of Biomedicine, Natural Products and Pharmacy)

Original Paper

Physico-chemical, sensory and microbial characteristics of fruit

flavoured milk based beverages during refrigerated storage.
Massarat Hassan1*, Basharat N. Dar1, Sajad A. Rather2, Rehana Akhter2, Aamina B. Huda1
1. Department of Food Science and Technology, IUST, Awantipora, Kashmir, India.
2. Department of Food Science and Technology, University of Kashmir, Srinagar, India-190006

*corresponding author email; [email protected]; Tel.: +91-9622660680

Running Title: Fruit flavoured milk based beverages

Received: 08 January, 2015; Accepted: 11 February, 2015

Abstract
Beverages based on fruit and milk products are currently receiving considerable attention as their market potential is growing.
Soft drink industry had made significant progress during recent years in terms of production, but there is only a limited range of
flavours available in India. The present study was carried out to develop novel milk based fruit flavoured beverages using 10, 15
and 20% apple juice (v/v) and sodium alginate as stabilizer (0.5%), in the formulation. The beverages were evaluated for their
stability and various physico-chemical, sensory and microbial properties during 7 days refrigerated storage. The pH value of the
beverages decreased from 6.3 - 6.0, 6.4 - 6.20 and 6.5 - 6.4; ascorbic acid content from 1.550 - 1.503%, 3.172 - 3.166% and
3.760 - 3.360% and total sugar content from 10.83 - 10.80%, 10.77 - 10.75% and 10.75 - 10.73% for T1, T2 and T3, respectively.
Whereas acidity increased from 0.117 - 0.282%, 0.225 - 0.290% and 0.273 - 0.295% and reducing sugars content from 4.49 -
4.55%, 4.40 - 4.42% and 4.34 - 4.39% for T1, T2 and T3, respectively. During refrigerated storage no significant difference (p
≤ 0.05) was observed with respect to fat, protein, viscosity and specific gravity but, total plate count showed a slight increase in
microbial count. The studies revealed that the milk based fruit flavored beverage prepared with 80% milk and 20% apple juice
scored maximum for sensorial quality attributes such as color, flavor and overall acceptability.

Keywords: Apple juice, Milk, Beverage, Stabilizers, Refrigerated storage.

Introduction particularly useful in places where, there is improper food and

nutrition leading to the deficiencies of some nutrients [1].
Beverages based on fruits and milk products are currently Most of the people do not relish milk and milk products as
receiving considerable attention as their market potential is such, so various flavoured milks have been made by using
growing. Soft drink industry had made significant progress variety off flavours like chocolate flavoured and fruit
during recent years in terms of production, but there is only a flavoured milk drinks.
limited range of flavours available in India. Many types of
syrups, sherbets and soft drinks containing artificial fruit Fruit and fruit products are rich source of many vitamins,
flavours are well known throughout the world. The basic essential minerals, digestible carbohydrates and organic acids
consideration is the caloric and therapeutic values, which that stimulate appetite, help in proper digestion and impart
make them popular and acceptable. At present fruit beverages specific flavour of foods. Milk based beverages are functional,
are generally synthetic flavoured, bottled and sold in the in that they provide all essential and non-essential amino acids
market. If this could be substituted with milk/whey, it will be but, are less acidic than fruit juices [2]. Nowadays new trend
more beneficial to consumers, dairy industries and beverage in beverage consumption is, increase in per capita carbonated
manufacturers. Beverages based on whey/milk have high soft drink consumption and a decrease in fluid milk
nutritional quality and increased caloric value. These could be consumption [3, 4]. Mixed fruit juice and milk beverages are

32
Fruit flavoured milk based beverages Massarat H et al

considered among the most functional and nutritional foods for 10 min and then kept undisturbed for a while. The
[5].Thus the beverage industry should incorporate new beverages were then filled in 200 ml capacity bottles which
ingredients in milk beverages which will improve their were already sterilized. The bottles were then crown capped
nutritional quality and organoleptic characteristics. by crown capped machine (Bajaj Moschinen Pvt. Ltd.) and
were pasteurized at a temperature of 70 – 75°C for 15- 20 min.
Maharaji apple is one of the low grade apple produced in
The beverages were then stored at refrigerated temperature for
Jammu and Kashmir, India. Its flesh is crisp, juicy, acidic and
further analysis.
aromatic. The variety sweetens on storage and has an excellent
shelf life. Juice of the fruit is prepared by mechanically Table 1: Basic formulation for the beverages with varying
squeezing or macerating fruit flesh without the application of apple juice concentrations
heat or solvents. Apple juices are consumed for their perceived
a
health benefits due to significant amounts of phytochemicals Ingredients Treatments
such as carotenoids, flavonoids, isoflavonoids, and phenolics
T1 T2 T3
[6]. Since apple juice is rich in antioxidants, a diet with these
components prevents oxidative stress, and may therefore Milk (ml/l) 900 850 800
reduce chronic diseases and slow aging [6]. The objective of
this study was to produce apple juice flavoured milk based Juice (ml/l) 100 150 200
beverage to give it apple flavour and to study its stability
during refrigerated storage. Sugar (g/l) 5 5 5

Materials and Methods Stabilizer 5 5 5

(g/l)
Materials
a
Fresh milk and apples were procured from the local market of Treatments: T1: containing apple juice (10%); T2: containing
Awantipora, Jammu and Kashmir, India. The damaged and apple juice (15%); T3: containing apple juice (20%).
rotten apples were rejected and only fresh and healthy apples Proximate composition
were used for juice extraction. The Stabilizer (sodium
Protein (991.20) and fat (920.85) contents were determined
alginate) and Sugar (sucrose) and chemicals used in this study
according to Association of Official Analytical Chemists,
were of analytical grade and procured from Himedia Pvt. Ltd.
AOAC [7]. The samples were analyzed in triplicate for each
Mumbai, India.
component.
Extraction of juice
pH and total titratable acidity
The apples were rinsed under running tap water, cut into small
The pH was determined with the help of electronic digital pH
pieces and crushed. The crushed apples were then pressed in
meter (LABINDIA). Buffer solutions of pH 4 and 7 were used
hydraulic press (Bajaj Moschinen Pvt. Ltd.) and the juice was
to calibrate the pH meter. Electrode was immersed in sample
extracted and filtered 2 to 3 times through a sterilized muslin
taken in a beaker, to determine pH. Total acidity was
cloth and then collected in glass beakers having a capacity of
determined by standard method of [8].
one liter. The juice was then pasteurized in an open pan and
was filled in bottles.
Formulation of fruit flavoured milk based beverages Total soluble solids
Three types of formulations were used for the preparation of The total soluble solids were determined by the standard
beverages in which the juice percentage varied from 10-20% method of [7] using hand refractometer (Atago Japan, DR- A-
1) at room temperature with a scale of 0-32°Brix.
(Table 1). The milk was filtered through sterilized muslin
cloth and was pre-heated to the temperature of 69 – 75°C. The
sugar (0.5%) and stabilizer (0.5%) were mixed in a mixer
(Philips) and added to milk at the temperature of 69 – 75°C.
The milk, stabilizer and sugar mixture was stirred for almost
15 minutes and was kept undisturbed for some time. The
mixture was then kept in a chiller (Philips, western), for ageing
over night (4°C or below). The juice was then added to the
milk at three different concentrations and was stirred in mixer
33
Fruit flavoured milk based beverages Massarat H et al

Milk Calculations:

Preheating (69 – 75ºC)

Addition of stabilizer-sugar mixture

Ageing of milk stabilizer-sugar mixture (4ºC or below,

over night) Specific gravity and viscosity
The specific gravity of the beverages was determined by
Mixing of juice (@ 10, 15, 20%) relative density bottle method. The empty relative density
bottle was weighed and filled with beverage (at 298K) and
weighed again (m). The procedure was then repeated for water
Stirring (ms) at 298K and specific gravity was calculated by the
following formula:

Bottling ρ/ρs = m/ms × V/Vs

The viscosity of the beverages was determined by Ostwald
Crown capping viscometer. The beverages were introduced into bulb A up to
mark, then forced under pressure into the bulb B. The
beverages were then allowed to fall freely back into the bulb
Pasteurization (70 – 75ºC) A and the time interval was recorded on stop watch (θ).The
procedure was repeated for the water and time was recorded
on stop watch (θ s). The values for the fluid viscosity were
Storing in refrigerator
determined by following relationship.
Scheme 1: Flow diagram showing the basic steps for the
preparation of fruit flavoured milk based beverages. µ =µs..θ /θs× ρ/ρs

Ascorbic acid Reducing sugars

Ascorbic acid content was determined according to [9]. The reducing sugars were determined by the standard method
Hundred milligram of L-Ascorbic acid was taken and the of [10]. The results were expressed as per cent on pulp weight
volume was made upto100ml with 3% hypochloric acid and basis.
then 10 ml was diluted to 100ml with 3% hypochloric acid. Microbial analysis
Dye solution was made by dissolving 50mg of sodium salt of
Total plate count was determined on nutrient agar media
2, 6-dichlorophenolindophenol in 150ml of hot glass water
according to [11]. Samples were prepared under aseptic
containing 42 mg of sodium bicarbonate and was diluted to
conditions. Ten ml from each sample was aseptically weighed
200 ml. Standardization of dye was done by taken 5ml of
into sterile tubes and blended for 2 min at high speed after
standard ascorbic acid solution and 5ml of 3% hypochloric
addition of 90 ml sterile 0.1% peptone water. Sevenfold serial
acid. The titration of this solution was done against the dye till
dilution was carried out. Each dilution was spread-plated, in
pink colour appears.
duplicate, on plate count agar (Hi Media Pvt. Ltd., Mumbai)
Dye factor=0.5/titrate used for enumeration. The inoculated petriplates were incubated at
37 ºC for 24 hour. Cfu/ml was calculated by taking average
Ten ml of sample was taken and volume made up to 100ml number of colonies which was multiplied by reciprocal of
with 3% hypochloric acid and was then filtered and dilution factor and expressed as Cfu/ml.
centrifuged. Finally 5 ml of aliquot was titrated against the dye
till pink color appeared.

34
Fruit flavoured milk based beverages Massarat H et al

Sensory evaluation 3.50% and 3.70-3.20% respectively. The T1 showed slight

increase, T2 did not show any change and T3 showed slight
The beverages were evaluated for different sensory
decrease in protein content, but the difference was not
parameters (colour, flavour, and overall acceptability) using
significant (p > 0.05). The fat content of the beverages ranged
9-point hedonic scale [12]. A selected ten member panel
from 1.53-1.45%, 1.60-1.55% and 1.80-1.79% respectively,
consisting of researchers and faculty members from the
and increased from treatment T1 to T3. There was slight non-
Department of Food Science and Technology, Islamic
significant decrease in fat content during refrigerated storage
University of Science and Technology, Awantipora. They
(p > 0.05) and the decrease in fat content could be due to the
were asked to express their opinion of the product. Samples
increase in juice percentage. Similar results were reported by
were evaluated for colour (9 =extremely desirable,
[13] in whey-based banana herbal (Mentha arvensis) beverage
1=extremely undesirable), flavour intensity (9 = extremely
during storage.
strong, 1 = extremely week to unpleasant) and overall
palatability (9 = palatable, 1 = unpalatable) using a 9-point As indicated in Table 2, acidity of the beverages ranged from
hedonic scale. Each attribute was discussed and tests were 0.117-0.282%, 0.225-0.290% and 0.273-0.295%, respectively
initiated after panelists were familiarized with the scales. Each and overall mean acidity value was found to be 0.205% and
sample was coded with a randomly selected 3-digit number 0.289% during refrigerated storage. Increase in titratable
and served in white cups. Panelists were instructed to cleanse acidity might be due to the growth of micro-organisms in
their palates between samples using cold water. beverages during storage or due to the conversion of lactose
into lactic acid by ascorbic acid present in the juice. The
Statistical analysis
conversion of proteins into amino acids could also be the
The data obtained was analyzed according to statistical reason for increased acidity during storage. The increase in
procedure of Analysis of Variance (ANOVA) using SAS 9.1 acidity from treatment T1 to T3 could be due to higher
software for Windows and the treatment means were percentage of juice content used in the basic formulation
computed using Least Significant Difference (LSD)at 5% (Table 1). The results obtained are conformity with those of
level of probability. earlier studies of [1], who worked on the development and
storage studies of whey based RTS beverage from mango cv.
Results and Discussion
kesar. Also similar results were found by [14] in studies on
Physico-chemical parameters development and storage of whey based pineapple
(Ananascomosus) and bottle gourd (Langenariasiceraria)
The physico-chemical parameters of fruit flavoured milk
mixed herbal beverage.
based beverages with varying fruit juice levels (10%, 15% &
20%) during refrigerated storage are presented in Table 2.
.
During refrigerated storage the mean protein content of
beverages (T1, T2 & T3) ranged from 3.45-3.69%, 3.50-

Table 2: Effect of refrigerated storage on physico-chemical composition of fruit flavoured milk based beverages
a
Storage Period Treatments Acidity (%) pH Protein (%) Fat (%)
0 days T1 0.117±0.14 6.3±0.11 3.45±0.06 1.53±0.11
T2 0.225±0.29 6.4±0.29 3.50±0.08 1.60±0.09
T3 0.273±0.18 6.4±0.07 3.70±0.06 1.80±0.13
Mean 0.205±0.23 6.36±0.20 3.55±0.10 1.64±0.12
7 days T1 0.282±0.23 6.0±0.14 3.69±0.11 1.45±0.07
T2 0.290±0.16 6.2±0.18 3.50±0.09 1.55±0.11
T3 0.295±0.12 6.5±0.12 3.20±0.06 1.79±0.11
Mean 0.289±0.21 6.20±0.19 3.46±0.10 1.60±0.09
LSD(P≤0.05) 0.002 0.11 N.S N.S

a
Treatments: T1: containing apple juice (10%); T2: containing apple juice (15%); T3: containing apple juice (20%).
35
 Adv. Biomed. Pharma. 2:1 (2015) 32-39

The pH values of the beverages ranged from 6.3-6.0, 6.4- Ascorbic acid
6.2 and 6.4-6.5, respectively and overall mean pH value was The ascorbic acid content of the fruit flavoured milk based
found to be 6.36 and 6.20 during refrigerated storage. beverages ranged from 1.503-1.550 mg/100ml, 3.172-3.166
During refrigerated storage there was significant decrease mg/100mland 3.760-3.360mg/100ml and overall mean
The pH value of the beverages ranged from 6.3 - 6.0, 6.4 - ascorbic acid content during refrigerated storage period was
6.2 and 6.5 - 6.4 respectively and overall mean pH value 2.811mg/100ml and 2.692mg/100ml (Figure. 2).
was found to be 6.36 and 6.20 during refrigerated storage. Decreasing trend was found in ascorbic acid content and
could be due to the auto-oxidation and light during
During refrigerated storage there was significant decrease in
refrigerated storage.[1], also reported decline in ascorbic
pH value and the decrease in pH might be due to slight acid content in whey based RTS beverage from mango cv.
growth of micro-organisms and their metabolites. Decrease kesar during storage, also similar results were found by [14],
in the pH may also be due to production of organic acids in studies on development and storage of whey-based
and amino acids due to the action of ascorbic acid on sugar pineapple (Ananascomosus) and bottle gourd
and protein content of the beverages. Similar results were (Langenariasiceraria) mixed herbal beverage.
reported by [15], in shelf life study of an orange juice and 0 day 7 days
milk based beverage after PEF and thermal processing and 4

[14], in studies on development and storage of whey based

3.5
pine apple (Ananascomosus) and bottle gourd
(lagenariasiceraria) mixed beverages. Also similar results 3

Ascorbic acid
were found by [16], in milk drink prepared by UHT
2.5
technique.
2
Total soluble solids
1.5
The mean TSS of 10%, 15% and 20% fruit flavoured milk
based beverages ranged from 14.8 – 15°Brix, 14.0- 1
14.2°Brix and 14.0-14.5°Brix, respectively (Figure 1). T1 T2 T3
Treatments
During storage increase in TSS (°Brix) content was found
and might be due to the solubilization of insoluble proteins Figure 2: Ascorbic acid content (mg/100ml) of the
due to presence of acids (ascorbic acid). Similar results were beverages during refrigerated storage.
reported by [14], who studied on development and storage
Reducing sugars and total sugars
of whey based pine apple (Ananascomosus) and bottle
gourd (Langenariasiceraria) mixed herbal beverage. Also The results of the reducing sugar content of the beverages
similar results were found by [16]. in milk drink prepared are shown in Figure 3. The reducing sugar content of the
by UHT technique. beverages ranged from 4.49-4.55%, 4.40-4.42% and 4.34-
4.39%, respectively. Increasing trend in reducing sugar
0 day 7 days
content was observed during refrigerated storage. Increase
15.2
in reducing sugar content might be due to the conversion of
15 sugar into reducing sugars in presence of citric acid. The
14.8
results are in agreement with those of [1]. who worked on
the development and storage studies of whey based RTS
TSS ( oBrix)

14.6
beverage from mango cv. Kesar.
14.4
The total sugar content of the beverages is presented in
14.2 Figure. 4. The data revealed that total sugar content of fruit
flavoured milk based beverages ranged from 10.83-10.80%,
14
10.77-10.75% and 10.75-10.73%, respectively. During
13.8 storage the total sugar content decreased and it might be due
T1 T2 T3
to the conversion and breakdown of high molecular weight
Treatments
polysaccharides. Similar results were obtained by [1], who
Figure 1: TSS (°Brix) of the beverages during worked on the development and storage studies of whey
refrigerated storage. based RTS beverage from mango cv. kesar.

36
 Fruit flavoured milk based beverages Advances in Biomedicine and Pharmacy Vol. 2 (1) 2015

Table 3: Effect of storage on Specific gravity and

0 day 7 days
viscosity of fruit flavoured milk based
4.6
beverages.
4.55
Storage Treatments Specific Viscosity (Pa s)
Reducing sugar (g/L)

4.5 Period Gravity

4.45 0 days T1 1.138±0.04 0.0076±0.03

4.4
T2 1.26±0.11 0.00278±0.02
T3 1.020±0.09 0.00126±0.02
4.35

Mean 1.130±0.10 0.0038±0.05

4.3
T1 T2 T3
7 days T1 1.140±0.13 0.0079±0.07
Treatments
T2 1.28±0.11 0.00289±0.09
T3 1.024±0.10 0.00127±0.10
Figure 3: Reducing sugar (%) content of beverages
during refrigerated storage Mean 1.183±0.14 0.00392±0.07
0 day 7 days LSD(P≤0) N.S N.S
10.84 a
Treatments: T1: containing apple juice (10%); T2:
10.82 containing apple juice (15%); T3: containing apple juice
(20%).
10.8
Sensory analysis
Total sugar

10.78
The results obtained in the sensory analysis are presented in
10.76
Table 4. The apple juice addition at different levels was
10.74 found to affect the sensory properties of fruit flavoured milk
10.72 based beverages. The results indicated that mean colour
score of the beverages during refrigerated storage ranged
10.7
T1 T2 T3 from 7.35-7.00, 7.27-7.10 and 7.55-7.30, respectively.
Treatments Overall mean colour score of the beverages was
Figure 4: Total sugar (%) content of beverages during 7.39and7.13. The colour score decreased with increase in
refrigerated storage storage period. The colour deterioration might be due to the
auto-oxidation by light, browning and degradation of
Specific gravity and viscosity
ascorbic acid. Mean flavour score of the beverages ranged
The results of the viscosity and specific gravity are from 7.45-7.10, 7.35-7.21 and 7.40-7.00, respectively and
presented in Table 3. The mean viscosity values of fruit overall mean flavour score was 7.40 and 7.10. Decrease in
flavoured milk based beverages ranged from 1.138-1.140 Pa flavour was observed in beverages during refrigerated
s, 1.260-1.280 Pa s and 1.020-1.024 Pa s, respectively and storage. The overall acceptability score of the beverages
overall mean viscosity values were 1.130and1.183Pa s. ranged from 7.12-700, 7.30-7.20 and 7.77-7.00,
During storage the change in viscosity was not significant respectively and overall mean acceptability was 7.40 and
(p > 0.05). The mean specific gravity values of beverages 7.06. Decreasing trend in overall acceptability was found
ranged from 0.00760-0.00790, 0.00278-0.00289 and during refrigerated storage. Decreasing trend in overall
0.00126-0.00127, respectively. Overall mean specific acceptability might be due to the decrease in all other
gravity was 0.00380-0.00392. During storage the change in sensory parameters (colour, flavour, taste and aroma) during
specific gravity was not significant (p > 0.05). The results storage. The results of the sensory analysis are in agreement
of the specific gravity and viscosity are in agreement with with those of [14] who worked on development and storage
those of [17], in case of development of beverages using of whey based pine apple (Ananascomosus) and bottle
fruit juice pulp, separated milk and reconstituted skim milk. gourd (Langenariasiceraria) mixed herbal beverage.

37
 Fruit flavoured milk based beverages Advances in Biomedicine and Pharmacy Vol. 2 (1) 2015

Table 4: Effect of refrigerated storage on Sensory Table 5: Effect of refrigerated storage on total plate
attributes of beverages. count of beverages.

aTreatments
Storage Colour Flavour Overall
Period acceptability Parameters Storage Samples Mean LSD
Period (P ≤
T1 T2 T3 0.05)
0 days T1 7.35±0 7.45±0. 7.12±0.46
.54 98 Total Plate 0 days 1.91×104 2.00×104 2.09×104 2.00×104 N.S
Count
T2 7.27±0 7.35±0. 7.30±0.67 (Cfu/ml) 7 days 2.30×104 2.15×104 2.35×104 2.26×104 N.S
.44 54 a
Treatments: T1: containing apple juice (10%); T2:
T3 7.55±0 7.42±0. 7.77±0.35 containing apple juice (15%); T3: containing apple juice
.29 59 (20%).
Mean 7.39±0 7.40±0. 7.40±0.64 Conclusion
.39 58
The results of this investigation revealed that apple juice can
7 days T1 7.00±0 7.10±0. 7.00±0.27
be used as flavouring agent for the production of flavoured
.21 65
milk based beverages. Fruit flavoured milk based beverages
T2 7.10±0 7.21±0. 7.20±0.38 were analyzed for acidity, pH, ascorbic acid, TSS, protein,
.43 92 fat, specific gravity, viscosity, reducing sugars and total
T3 7.30±0 7.00±0. 7.00±0.49 sugars. During refrigerated storage the beverages exhibited
.33 28 increasing trend in acidity, TSS and reducing sugar contents
Mean 7.13±0 7.10±0. 7.06±0.4 while as, decreasing trend was observed in pH, ascorbic acid
.47 31 content and total sugars. The parameters like fat, protein,
LSD(P ≤ 005) 0.09 0.07 0.05 viscosity and specific gravity did not show any significant
a
Treatments: T1: containing apple juice (10%); T2: change during refrigerated storage. The microbial analysis
containing apple juice (15%); T3: containing apple juice showed non-significant change in total plate count during
(20%). refrigerated storage. The studies revealed that fruit flavored
Microbial analysis milk based beverage prepared with 80% milk and 20%
apple juice scored maximum for sensorial quality attributes
The results obtained with respect to total plate count of the such as colour, flavor and overall acceptability. Thus apple
beverages are shown in Table 5. Total plate count of the juice at 20% concentration can be used as a substitute for
beverages during refrigerated storage ranged from synthetic flavours in milk beverages without any
1.91×104-2.30×104, 2.00×104-2.15×104 and 2.09×104- detrimental effects to the quality of the product as perceived
2.35×104,, respectively and overall means values were by the consumer.
2.00×104 and 2.26×104. The microbial count of the
beverages incorporated with 10%, 15% & 20% apple juice
did not differ significantly from 0 to 7 days during
Conflict of interest
refrigerated storage (p > 0.05). The microbial level detected
(104 cfu/ml) is within the normal range observed in this type The authors declare that there is no conflict of interest
of product during 0 day to 7 days refrigerated storage. The related with this publication.
results obtained are in agreement with those of [1], who
worked on the development and storage studies of whey
based RTS beverage from mango cv. Kesar, also similar
results were found by [15], in shelf life study of an orange
juice and milk based beverage after PEF and thermal
processing.

38
Fruit flavoured milk based beverages Advances in Biomedicine and Pharmacy Vol. 2 (1) 2015

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