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Development of Whey Protein Beverage from Mozzarella Cheese Whey

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Proceedings of National Conference on Food Science and Technology (Food Conference-2012), 10-11 August, 2012, Kathmandu, Nepal

Development of Whey Protein Beverage from

Mozzarella Cheese Whey
Sarala Prajapati1*, Pravin Ojha1 and Ishwar Subedi2
National College of Food Science and Technology, Khusibu, Kathmandu
1

2
Department of Food Technology and Quality Control, Babarmahal, Kathmandu

Whey protein was precipitated from Mozzarella cheese whey by concentrating at 90 oC for 10 min
at different pH. The maximum protein concentrate was achieved at a pH of 3.5 with a yield of 4.3
± 0.1%. Furthermore, whey protein beverage was developed using different proportions of whey
protein. It was found that the beverage made from 4% protein had the best sensory qualities. Shelf
life studies of the beverage packed in glass bottles were done under refrigerated conditions for 60
days. The results revealed that there was a slight change in the acidity of the beverage. Microbial
analysis showed an increase in the total plate count and mold count during the storage period,
however, with insignificant effects on the overall quality of the product. It seems that whey protein
beverage could be developed from mozzarella cheese whey and could be stored for 60 days under
refrigerating conditions.

Keywords: Cheese whey, Whey protein, Protein concentrate, Beverage

Introduction
Whey, the yellow-green liquid that separates from the curd during manufacture of cheese and
casein, has long been considered by the dairy to be a waste byproduct and, thus a disposal problem.
Whey is a source of biological and functional valuable protein i.e. whey protein (Legarova and
Kourimska, 2010). Milk contains two major protein fraction, including casein, which provides
about 80% by weight of the total protein and whey protein, which provides about 20% by weight
of the total protein. The proteins appearing in the supernatant of milk after precipitation at pH 4.6
are collectively called whey proteins. These globular proteins are more water soluble than caseins
and are subject to heat denaturation. The principle fractions are ß-lactoglobulin, alpha-lactalbumin,
bovine serum albumin (BSA), and  immunoglobulin (Ig) and the minor proteins are lactoferrin,
glycomacropeptide, lactoperoxidase (Acharya, 2010). Whey protein is a pure, natural, high quality
protein of milk. It is one of the best proteins for human food use, due to its balanced amino acid
profile and superior digestibility (Adhikari, 2005). Whey protein contains all of the essential
amino acids, and therefore, is a high quality, “complete source” of protein (Luhovy, 2007). More
specifically, whey protein are a rich source of branched chain amino acid (BCAAs), containing the
highest known levels of any natural food source (Etzel, 2004). BCAAs are important for athletes,
since, unlike the other essential amino acid, they are metabolized directly into muscle tissue and
are the first amino acid used during periods of exercise and resistance training (Jelicic et al., 2008).

Whey proteins are often the preferred source for ready-to-drink (RTD) protein beverages because
of their excellent nutritional qualities, bland flavor, ease of digestibility, and unique functionality
in beverage systems (Rittmanic, 2006). Whey proteins are added to a variety of foods and
beverages for functionality and added nutrition. A rapidly growing area of whey protein use in
foods and beverages is the sports drink category. There are two categories of whey protein-fortified
drinks: those at neutral pH and those at low pH. The drinks at low pH have a clear and refreshing
appearance, compared to the shake-style drinks at neutral pH (Beecher, 2006).

*Corresponding author, E-mail: salucall@gmail.com 123

Proceedings of National Conference on Food Science and Technology (Food Conference-2012), 10-11 August, 2012, Kathmandu, Nepal

Most of the cheese industry of Nepal are throwing the whey as the waste and not attempting to
utilize it in new product. By utilizing whey this industries not only produce useful product but also
reduce their cost of main product i.e. cheese. Therefore the attempt has been made to utilize the by-
product of cheese industry (i.e. whey) into a useful product such as a whey protein and incorporate
it in the formulation of ready-to-drink beverage.

Materials and Methods

The whey used for precipitation of whey protein concentrate was Mozzarella cheese whey produced
at Nepal Dairy Pvt. Ltd, Hattiban, Lalitpur. Firstly cheese whey was collected in plastic jars (5
liters) and then immediately stored at refrigeration temperature 40C in industry. Then the whey
was transported at the chilling condition from the industry to the laboratory. And then proximate
analysis (pH, acidity, fat, protein, lactose, total ash, and total soluble solid) of whey was carried out.
Filtration of whey was done in order to remove remaining solid parts of cheese before precipitation
of whey protein. After that three different precipitation methods were carried out to isolate whey
protein concentrate (WPC) and thus precipitated WPC was freshly incorporation in formulation of
ready-to-drink beverage (i.e. whey protein beverage).

Method-A: This is heat precipitation method propose by Matthews (1984). In this method, cheese
whey was directly heated to temperature 900C for 10 minutes at its original pH. During heating
whey protein was precipitated and cooled to room temperature. Then it was centrifuged at 5000
rpm for 15 minutes to separate whey protein concentrate and qualitative test (protein content and
solubility) of separated WPC was done.

Method-B: This is also heat precipitation method but it is slightly different than previous method.
This method was proposed by El. Desoki, (2009). In this method, first pH of whey was adjusted
to 4.6 and then heated to temperature 900C for 10 minutes. During heating whey protein was
precipitated and cooled to room temperature then left for overnight. Next supernatant was thrown
out and then centrifuged at 5000 rpm for 15 minutes to separate whey protein concentrate and
qualitative test (protein content and solubility) of separated WPC was done.

Method-C: This is also heat precipitation method but it is slightly different than previous two
methods. This method was proposed by Modler and Emmons (1976); Lupano (1994). In this
method, first pH of whey was adjusted to 2.0-3.5 and then heated to temperature 900C for 10
minutes. After that it was cooled up to 60-550C then 2% Sodium bicarbonate (NaHCO3) was added
up to pH 4.5 to precipitate whey protein and then it was left for overnight. Next centrifugation was
done at 5000 rpm for 15 minutes to separate whey protein concentrate (WPC) and qualitative test
(protein content and solubility) of separated WPC was done.

Whey protein beverage: The freshly precipitate whey protein concentrate (WPC- 45%) from
cheese whey was used for the production of whey protein beverage. The beverage was prepared
by adding WPC in processed water and 10% sugar was added in it. It was then mixed well so
that all sugar get dissolved in the solution then pH of beverage was reduced to 4.1 by adding 2%
ortho-phosphoric acid. Then it was heated up to 720C for 1 min and 0.3% cardamom flavor, 0.01%
apple green color and 0.04% potassium sorbate were added to it before bottling in sterilized glass
bottle. After that the bottle was sealed with cap and stored at 40C immediately. Optimization of
product was carried out by developing beverage with WPC at different proportion i.e. 1%, 2%,
3% and 4% and sensory evaluation was done to get the best final product in term of color, flavor,

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Proceedings of National Conference on Food Science and Technology (Food Conference-2012), 10-11 August, 2012, Kathmandu, Nepal

taste, appearance, mouth-feel and overall acceptability. The shelf life of prepared beverage filled
in sterilized glass bottle and stored at refrigerated condition (40C) was done for 60 days in terms of
chemical parameters (pH and acidity) and microbial count.

Analytical procedure- While examining fresh whey and final product; whey protein beverage,
only few parameters like total soluble solids by Hand Refractometer (Ranganna, 2001), fat content
by Gerber Method (AOAC, 2005), protein content by Kjeldahl Method (AOAC, 2005), acidity
by titration method (Pearson, 1981), pH by pH meter, total sugar and lactose content by Lane and
Enyon method (AOAC, 2005), total ash content by electric muffle furnace (Ranganna, 2001) were
analyzed.

Microbial analysis of final product (such as Total plate count, Total coliform count and Yeast and
mold count) was done by Pour plate technique (NDDB, 2001).

Sensory evaluation- Sensory evaluation was performed by 9 point hedonic scoring test (9 =
like extremely, 1 = dislike extremely) for color, flavor, taste, appearance, mouth-feel and overall
acceptance. The evaluation was carried out by 10 panelists comprising of graduate students,
teachers and staffs of NCFST. Each panelist was provided with 4 samples coded with random
numbers for final product with Sensory evaluation card.

Statistical analysis- Data on physicochemical and sensory analysis were tabulated for comparison
and were graphically represented using Microsoft Excel-2007, Data were statistically processed
by GenStat Discovery Edition 4, GenStat for Analysis of Variance (ANOVA). Means of the data
were separated whether they are significant or not by using LSD (least square difference) method.

Results and Discussions

The whey used for the production of whey protein concentrate (WPC) was Mozzarella cheese
whey. The results obtained from proximate analysis of fresh cheese whey are listed below in
tabulated form in Table 1.

Table 1. Physio-chemical composition of fresh cheese whey

Parameters Amounts
Lactose (%) 4.5±0.51
Protein (%) 0.9±0.03
Fat (%) 0.5±0.14
Total ash (%) 0.42±0.01
Moisture content (%) 93.02±0.48
TSS (0Bx) 6.0
pH 4.2 at 250C
Acidity (%) 0.40±0.02 (as lactic acid)

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Proceedings of National Conference on Food Science and Technology (Food Conference-2012), 10-11 August, 2012, Kathmandu, Nepal

Table 2. Yields and protein content from different methods

Methods Yields (%) Protein Contents (% db) Protein Solubility

A 4.01a ±0.29 38.40a ±0.62 Not good

B 3.88a ±0.32 41.98b ±0.99 Slightly good
C 3.65 ±0.21
a
45.10 ±0.52
c
Very good

Note: Values are mean ± s. d. Values followed by same superscripts within same column are not
significantly different (p<0.05) and different superscripts within same column are significantly
different (p<0.05)

Table 2 showed that the yield from the method A was highest (i.e. 4.01±0.29%) than other two
methods but protein content (i.e. 38.40±0.62%) and its solubility was relatively lower than
other methods. The protein content of whey protein from method C was found to be highest
(45.10±0.52%) and the solubility of protein was very good than other methods even though the
yield 3.65±0.21% from this method was less. Thus method C was selected as the best method
for the further precipitation of whey protein concentrate (WPC) on the basis of protein content
and its solubility. The statistical results showed that there was no significant difference in yields
(p<0.05) between methods A, B and C but significant difference was found in protein content
(p<0.05) between all the three methods. The yield of whey protein was found to be affect by
pH and heat treatments during precipitation from different methods. The protein content of whey
protein concentrate (WPC) from method C was highest than other two methods. This might be due
to presence of low amount of lactose, fat and mineral content in WPC from method C.

Table 3. Yields of whey protein at different pH

pH Yields (%)
2 3.57a ±0.08
2.5 3.75b ±0.07
3 3.98c ±0.08
3.5 4.30d ±0.06

Note: Values followed by different superscripts within same column are significantly different
(p<0.05)

For the optimization of method C, whey protein was precipitated at different pH values i.e. 2.0, 2.5,
3, 3.5 and yield was obtained as in the above table 3. The yield of whey protein concentrate (WPC)
at pH 3.5 was found highest (i.e. 4.30±0.06%) and least was at pH 2 (i.e. 3.57±0.08%) than other
pH. Therefore precipitation process was optimized on the basis of its yields from experiments and
further precipitation of WPC was done at pH 3.5 for the incorporation in protein beverage. . The
statistical results showed that there was significant difference in yields (p<0.05) between all the
samples at pH value 2.0, 2.5, 3, 3.5. The mean score for the yield at pH 3.5 was found to be highest
(i.e. 4.313) and the least was at pH 2 (i.e. 3.540).

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Proceedings of National Conference on Food Science and Technology (Food Conference-2012), 10-11 August, 2012, Kathmandu, Nepal

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setartnecnoc nietorp yehw ehT .egareveb ni tnetnoc nietorp yehw fo sisab eht no dezimitpo saw
ytilibulos sti dna tnetnoc nietorp ,sdleiy fo sisab eht no )C dohteM( dohtem tseb morf etatipicerp
egareveb nietorp yehW .egareveb ni etartnecnoc nietorp yehw fo noitaroprocni eht rof desu saw
htiw retaw decnahne-nietorP fo epicer ro noitalumrof ni noitacfiidom eht htiw depoleved saw
etartnecnoc nietorp yehW .)8002 ,nonA( .cnI tnemeganaM yriaD yb nevig rovafl yrrebwarts larutan
nehT .egareveb nietorp yehw eht poleved ot %4 dna %3 ,%2 ,%1 fo noitroporp eht ni desu saw
eht hguorht deniatbo saw nietorp yehw fo noitroporp etairporppa eht htiw tcudorp lanfi tseb eht
.noitaulave yrosnes

erew snoitisopmoc lairt fo noitaulave yrosneS -egareveb nietorp yehw fo noitaulave yrosneS
.noitaulave yrosnes hguorht egareveb ni nietorp yehw fo noitroporp tseb eht tuo dnfi ot tuo deirrac
leef htuom ,ecnaraeppa etsat ,rovafl ,roloc fo sisab eht no detaulave erew depoleved stcudorp ehT
.1 erugiF eht ni nevig era noitaulave yrosnes yb deniatbo stluser ehT .ecnatpecca llarevo dna

egareveb fo setubirtta yrosnes no noitairav nietorp yehw fo tceffE .1 erugiF

%2 htiw tcudorP =B elpmaS ,)CPW( etartnecnoc nietorp yehw %1 htiw tcudorP =A elpmaS :etoN
CPW %4 htiw tcudorP =D elpmaS ,CPW %3 htiw tcudorP =C elpmaS ,CPW
.yllacitsitats )50.0 < p( tnereffid yltnacfiingis ton setacidni srab eht fo pot ta tebahpla emaS

elpmas neewteb )50.0<p( roloc ni ecnereffid tnacfiingis saw ereht taht dewohs sisylana lacitsitatS
dna etsat ,rovafl ni dnuof saw ecnereffid tnacfiingiS .erocs yrosnes fo mret ni D elpmas dna A
.erocs yrosnes fo mret ni D elpmas htiw C ,B ,A elpmas neewteb )50.0<p( ytilibatpecca llarevo
B elpmas ,D dna A elpmas neewteb dnuof saw )50.0<p( ecnereffid tnacfiingis ,leefhtuom roF
ecnaraeppa ni dnuof saw ecnereffid tnacfiingis on tuB .C dna B elpmas ,D dna C elpmas ,D dna
yehw %4 htiw tcudorp eht yllacitsitatS .erocs yrosnes fo mret ni selpmas eht lla neewteb )50.0<p(
sti fo lla naht roirepus )50.0<p( yltnacfiingis eb ot dnuof saw )D elpmaS .e.i( etartnecnoc nietorp
.strapretnuoc

tcudorp lanfi fo noitaulave yrosnes fo yrammus llarevO

A elpmaS > B elpmaS > C elpmaS > D elpmaS :roloC
C elpmaS > B elpmaS = A elpmaS > D elpmaS :rovalF
A elpmaS > B elpmaS > C elpmaS > D elpmaS :etsaT
A elpmaS > B elpmaS > C elpmaS =D elpmaS :ecnaraeppA

127
Proceedings of National Conference on Food Science and Technology (Food Conference-2012), 10-11 August, 2012, Kathmandu, Nepal

Mouthfeel: Sample D > Sample C > Sample B > Sample A

Overall acceptance: Sample D > Sample C > Sample B > Sample A

Therefore Sample D with 4% whey protein was selected as a best product from sensory evaluation.

Table 4. Proximate analysis of final product (Sample D)

Parameters Amounts
pH 4.1
Acidity (%) 0.13±0.03 (as phosphoric acid)
TSS ( Bx)
0
10.0
Fat (%) 0.3±0.02
Protein (%) 3.7±0.03
Total ash (%) 0.51±0.02
Total sugar (%) 12.0±0.33
Moisture content (%) 85.94±0.34

Finally product was prepared with recipe of 85% water, 4% whey protein, 10% sugar, 0.3%
cardamom flavor, 0.01% apple green color and 0.04% potassium sorbate. Table 4 showed the
results of proximate analysis of final product. According to Ismail et al., (2011), the result of
chemical analysis of Mango based whey beverage was pH 4.86 with acidity 0.15% as ascorbic
acid, TSS 140Bx and Total sugar 16%.

Shelf life of the product- Shelf life evaluation was carried out for the highest sensory scored
beverage sample (i.e. Sample D). It was stored in packaging material (glass bottle) at refrigeration
condition i.e. 40C. Shelf life of the product was evaluated according to chemical parameters (like
acidity and pH) and microbial count (such as total plate count, total yeast and mold count and
coliform count).

Figure 2. Microbial profile of final product during storage period

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Proceedings of National Conference on Food Science and Technology (Food Conference-2012), 10-11 August, 2012, Kathmandu, Nepal

The above graph showed that the microbial load was increased with the increase in storage period.
The TPC was found 0.2×103 cfu/ml even after pasteurization of product during packaging and then
it was found increase in load up to 2.9×103 cfu/ml on 60 days of storage. The increase in microbial
load was not so high that it affects the quality of product. The yeast and mold was absence during
packaging of product but it was found increase up to 2.0×103 cfu/ml on 60 days. The analysis
showed that the final product was found to be free of Coliform for 60 days during storage period.
The microbial limits for non-alcoholic beverage are TPC<50,000 cfu/ml and coliform must be
absent (Stannard, 1997). And the result obtained for TPC was within the limit and coliform was
absence in stored product for 60 days. Thus these conclude that the product was acceptable even
after 60 days storage at low temperature.

Figure 3. Change in pH and acidity of final product during storage period

The pH and acidity of the final product was determined for 60 day at the interval of 15 days during
storage period at refrigerated condition. From the above graph showed that the pH and acidity of
the product was found constant i.e. pH 4.10 and 0.13% acidity up to 15 days and started decreasing
pH from 4.10 to 4.00 and increasing acidity from 0.13 to 0.19% after 15 days storage. There was no
significant change found in pH and acidity of product. And there was not seen any affect on quality
of product even after storage for 60 days at low temperature. These results were in agreement with
the findings of Ismail et al., (2011), who reported a decrease in pH and an increase in acidity of
whey-based mango beverage during storage at low temperature (i.e. 40C). According to Hussain
et al., (2010), the decrease in pH and increase in total titratable acidity during cold storage period
may be due to activity of some acid producing bacteria such as Alicyclobacillus acidoterrestris and
due to chemical reactions.

Table 5. Energy value per 100 ml of whey protein beverage

Nutrients Quantity (g) Energy/g Total energy (Kcal)
Total carbohydrate 12 4 48
Total protein 3.7 4 14.8
Total fat 0.3 9 2.7
Grand total 65.5

Table 5 showed the energy value per 100 ml of final product. Hence, energy contributed by 100ml
of prepared whey protein beverage was found to be 65.5 Kcal.

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 Proceedings of National Conference on Food Science and Technology (Food Conference-2012), 10-11 August, 2012, Kathmandu, Nepal

Conclusion
The study showed that whey protein concentrate precipitated from method C proposed by Lupano,
(1994); Modler and Emmons, (1976) was found to be best on the basis of protein content and its
solubility and the yield of whey protein was higher at pH 3.5. The 4% whey protein concentrate
beverage was more acceptable on the basis of sensory evaluation. Shelf life of the product packed
in glass bottle and stored at refrigerated condition (40C) was found to be better for 60 days on the
basis of microbiological analysis. Finally ready-to-drink whey protein beverage was developed
with 4% whey protein concentrate. Development of product with different flavors and fruit juices
and with or without the addition of minerals, vitamins and antioxidant on sensory quality of product
can be studied.

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