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Protein and Amino Acid Profiles of

Different Whey Protein Supplements
a

Cristine C. Almeida , Thiago S. Alvares , Marion P. Costa & Carlos A.

Conte-Junior

Department of Food Technology, Fluminense Federal University, Rio

de Janeiro, Rio de Janeiro, Brazil
b

Nutrition Institute, Federal University of Rio de Janeiro, Rio de

Janeiro, Brazil
Published online: 28 Aug 2015.

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To cite this article: Cristine C. Almeida, Thiago S. Alvares, Marion P. Costa & Carlos A. Conte-Junior
(2015): Protein and Amino Acid Profiles of Different Whey Protein Supplements, Journal of Dietary
Supplements
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Journal of Dietary Supplements, Early Online:111, 2015

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DOI: 10.3109/19390211.2015.1036187

ARTICLE

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Protein and Amino Acid Profiles of Different Whey

Protein Supplements
Cristine C. Almeida1 , Thiago S. Alvares2 , Marion P. Costa1 ,
& Carlos A. Conte-Junior1
1

Department of Food Technology, Fluminense Federal University, Rio de Janeiro, Rio de

Janeiro, Brazil, 2 Nutrition Institute, Federal University of Rio de Janeiro, Rio de
Janeiro, Brazil

ABSTRACT. Whey protein (WP) supplements have received increasing attention by

consumers due to the high nutritional value of the proteins and amino acids they provide. However, some WP supplements may not contain the disclosed amounts of the ingredients listed on the label, compromising the nutritional quality and the effectiveness
of these supplements. The aim of this study was to evaluate and compare the contents of
total protein (TP), -lactalbumin (-LA), -lactoglobulin (-LG), free essential amino
acids (free EAA), and free branched-chain amino acids (free BCAA), amongst different WP supplements produced by U.S. and Brazilian companies. Twenty commercial
brands of WP supplements were selected, ten manufactured in U.S. (WP-USA) and ten
in Brazil (WP-BRA). The TP was analyzed using the Kjeldahl method, while -LA, LG, free EAA, and free BCAA were analyzed using HPLC system. There were higher
(p < 0.05) concentrations of TP, -LA, -LG, and free BCAA in WP-USA supplements, as compared to the WP-BRA supplements; however, there was no difference
(p > 0.05) in the content of free EAA between WP-USA and WP-BRA. Amongst the
20 brands evaluated, four WP-USA and seven WP-BRA had lower (p < 0.05) values
of TP than those specified on the label. In conclusion, the WP-USA supplements exhibited better nutritional quality, evaluated by TP, -LA, -LG, and free BCAA when
compared to WP-BRA.
KEYWORDS.

amino acids, dietary supplement, -lactalbumin, -lactoglobulin

INTRODUCTION
Whey protein (WP) represents almost 2030% of the total protein in bovine
milk, which is a complex mixture of globular protein molecules consisting of lactalbumin (-LA), -lactoglobulin (-LG), and other minor proteins (Urista,

Alvarez,
Riera, Cuenca, & Tellez,
2011). The protein fractions -LA and -LG
(variants A and B) represent almost 70% of the proteins present in whey (Walstra,
Address correspondence to: Carlos Adam Conte-Junior, Rua Vital Brazil Filho, n. 64. Santa Rosa, CEP:

24.230-340, NiteroiRio
de Janeiro, Brasil (E-mail: carlosconte@id.uff.br)
(Received 08 September 2014; accepted 05 March 2015)

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Almeida et al.

Wouters, & Geurts, 2006). Both proteins are a valuable source for essential amino
acids, and are therefore seen as nutrients that provide important physiological benefits (Toro-Sierra & Ulrich, 2013). The WP supplements have been recognized
for their high nutritional quality, fast absorption, and as a rich source of essential
amino acids (EAA), mainly branched-chain amino acids (BCAA), such as leucine,

isoleucine, and valine (Almeida, Conte-Junior,

Silva, & Alvares, 2013).
In the last few years, the consumption of WP supplements has been recommended to stimulate muscle protein synthesis in physically active individuals,
mainly in individuals practicing resistance exercise (Phillips, 2012). In addition, the
scientific knowledge evidencing the relationship between physical performance,
and whey protein consumption stimulated the rapid expansion of new dietary supplements manufactured from these proteins (Borsheim, Tipton, Wolf, & Wolfe,
2002).
According to the Federal Trade Commission, the US consumer protection
agency, hundreds of dietary supplements exhibited irregularities recently, including lower quantities of protein than the disclosed values (FTC, 2013). Furthermore, recent analysis of WP supplements raised concerns in both the United States
and Brazil because some of these products contained high carbohydrates and/or
low protein contents than those listed on the ingredients label (Moore, Spink, &
Lipp, 2012; ConsumerReport, 2010). As a result, the Brazilian Health Surveillance
Agency (ANVISA) suspended the sale of some batches of protein supplements
due to carbohydrates contents greater than the 20% (w/w) limit (ANVISA, 2014).
These observations could be suggestive of lower nutritional quality of the ingredients used (i.e. addition of proteins of low biological value, such as vegetable proteins), the addition of other nonprotein ingredients (i.e. carbohydrates), or misleading label description.
Considering the high demand from the physically active population for the use of
WP supplements, consumers expect that such products contain a minimum amount
of nutrients of high biological value, necessary to promote the physiological benefits. For these reasons, the aim of this study was to evaluate and compare the contents of total protein (TP), -LA, -LG, free EAA, and BCAA, in different brands
of WP supplements produced by U.S. companies and by Brazilian companies. Furthermore, it was compared the TP content of each WP supplements and the TP
value informed on their respective labels.

MATERIAL AND METHODS

Sample Selection
The supplements used in the present study were acquired through an online store
specialized on nutritional supplements. Twenty WP supplements were selected
from their bestseller list, where 10 WP supplements (WP isolated: n = 5 and WP
concentrated and isolated: n = 5) were manufactured by U.S. companies (WPUSA) whereas 10 WP supplements (WP isolated: n = 5 and WP concentrated:
n = 5) were manufactured by Brazilian companies (WP-BRA). All products were
within their shelf-life ranging their expiration date between November 2014 and
June 2015.

Protein and Amino Acid Analysis in Supplements

Total Protein Analysis

The total protein content was evaluated according to the Kjeldahl method and the
corresponding nitrogenous materials were converted into % of protein, by multiplying by the factor of 6.38, according to the AOAC (2012) methods. The analysis
was performed in quintuplicate.

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Protein Fractions Analysis

A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was
performed to visualize the protein profile in the WP supplements. The samples
were prepared according to the method described by Criscione et al. (2009), with
some modifications, followed by the method developed by Bradford (1976) to estimate the approximate protein concentration in the samples. The stacking gel
and resolution gel were 4% and 10% of acrylamide, respectively. The gel was run
(120 V, 25 mA for approximately 2 h) after full polymerization and application
of 20 L of sample (5 mg/mL of protein) in each well. After electrophoresis, gels
were stained with Coomassie Blue solution G-250 for 24 h and then destained with
a solution of 20% acetic acid, 20% methanol, and 60% distilled water until the
background was completely clean (Conte-Junior, Golinelli, Paschoalin, & Silva,
2006). The destained gels were photographed and the data were evaluated by ToR
software to estimate band density. The apparent molecular weights
talLab Quant
were estimated using electrophoresis protein standard (Precision Plus Protein Dual
Color Standards, Bio-Rad).
The quantification of major protein fractions, -LA and -LG (variant A and
variant B), were analyzed by high-performance liquid chromatography (HPLC)
system. The protein extraction followed the protocol as proposed by Bobe, Beitz,
Freeman, and Lindberg (1998). Before beginning the extraction procedure, all samples were diluted according to the manufacturers recommendation. After this,
500 L of each sample was mixed with 500 L of a solution containing 0.1 M
BisTris buffer, 6 M Guanidine hydrochloride, 5.37 mM sodium citrate and 19.5 mM
d-Dithiothreitol. Each sample was shaken during 10 s and incubated during 1 h at
room temperature, and then diluted to 1:3 (v:v) with a solution containing 4.5 M
Guanidine hydrochloride. Finally, the sample was filtered through a PVDF membrane of 0.22 m.
The HPLC system consisted of a quaternary pump (LC-20AD, Shimadzu Corporation, Japan), analytical column Zorbax 300SB-C8 (4.6 mm ID 250 mm Agilent
Technologies), and a photodiode array detector (SPD-20A, Shimadzu Corporation, Japan). Elution was carried out at flow rate of 0.5 mL.min1 using a mixture
of two solvents: solvent A consisted of 0.1% trifluoroacetic acid (TFA) in water and
solvent B was 0.1% TFA in acetonitrile. The total analysis time for each sample was
45 min. The column temperature was kept at 45 C and the detection wavelength
was set at 214 nm. (Bonfatti, Grigoletto, Cecchinato, Gallo, & Carnier, 2008).
The -LA, -LG variant A and -LG variant B external standards were dissolved with a solvent containing acetonitrile, water, and TFA in a ratio of 100:900:1
(v:v:v) to the final concentrations of 12.5 mg.mL1 . Calibration curve was plotted
utilizing six data points: 12.5; 6.25; 3.12; 1.56; 0.78; 0.39 mg.mL1 . The samples and
standards were conducted in triplicate.

Almeida et al.

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Essential Amino Acids (EAA) Analysis

The free EAA (histidine, threonine, methionine, valine, phenylalanine, isoleucine,
leucine, and lysine) were analyzed as described by Alvares et al. (2012). Before
analysis, all samples were diluted as recommended by the manufacturer. Fifty L
of diluted sample was mixed with 50 L of 1.5 M HClO4 in a 1.5 mL microcentrifuge tube. After 2 min at room temperature, 1.125 mL ultrapure water and 25 L
of 2 M K2CO3 were added. The tubes were vortexed and centrifuged at 10,000 g
for 1 min in a microcentrifuge, a volume of 100 L of the supernatant was diluted
with 100 L of 1.2% benzoic acid and 1.4 mL DD-water, and then 50 L of this
solution was mixed with 50 L of the o-phthaldialdehyde (OPA) reagent solution
(v:v) for 1 min and 50 L was immediately delivered into the HPLC. The HPLC
system was equipped with a quaternary pump (LC-20AD, Shimadzu Corporation,
R
)
Japan), a 5 m reversed-phase C18 column (4.6 mm ID 150 mm from Supelco

R
guarded by a 5 m reversed-phase C18 guard column Ascentis (4.0 mm ID
R
) and a fluorescence detector (RF-10AXL, Shimadzu Cor20 mm from Sigma
poration, Japan) monitoring excitation and emission wavelengths at 340 nm and
455 nm, respectively. The samples were separated by gradient using 0.1 M sodium
acetate (pH 7.2) and methanol as mobile phase at a flow of 1.1 mL min1 . The total
running time per sample was 49 min and the column temperature was kept at room
temperature.
Statistical Analysis
One sample t-test was used to identify differences in the content of TP between the
brands of WP supplements with the content of TP informed in the respective label.
A MannWhitney test for independent sample was used to identify differences in
TP, -LA, -LG, free EAA, and free BCAA between the USA and BRA WP
supplements. Statistical significance was set at the 95% of confidence level. All
analyses were performed using a commercially available statistical package (IBM
SPSS Statistics version 22 for Mac, Chicago, IL), and the results were expressed as
means SD.
RESULTS
Total Protein
The content of total protein of the WP-USA (72.83 5.8 %) and WP-BRA (63.36
8.4 %) supplements are presented in the Figure 1. There was difference (p <
0.05) in TP content between the WP-USA and WP-BRA supplements.
Among the 10 WP-USA brands evaluated, the TP content of the 4 brands was
lower (p < 0.05) that the values described respective labels (WP-USA a: 61.9%
vs. 74.3%; WP-USA b: 76.6% vs. 84.0%; WP-USA d: 79.6% vs. 83.3%, and WPUSA e: 69.9% vs. 81.7%). Regarding the 10 WP-BRA supplements, 7 WP-BRA
supplements were lower (p < 0.05) in TP content than their respective label values
(WP-BRA a: 75.3% vs. 91.7%; WP-BRA b: 62.0% vs. 90.0%; WP-BRA d: 67.7%
vs. 88.7%; WP-BRA e: 58.8% vs. 80.0%; WP-BRA f: 72.4% vs. 80.0%; WP-BRA
g: 60.9% vs. 70.0%; WP-BRA h: 62.0% vs. 69.7%).

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Protein and Amino Acid Analysis in Supplements

FIGURE 1. Total protein (%) of WP-USA (A) and WP-BRA (B) supplements. TP = total
protein; WP-USA = whey protein supplements produced by U.S. companies; WP-BRA =
whey protein supplements produced by Brazilian companies. The symbol (p < 0.05)
denotes significantly different from WP-BRA.

Protein Fractions
The protein profile observed in the SDS-PAGE demonstrated that the most expressive bands were located in the region with a molecular weight of around 10
and 19 kDa, representing the apparent molecular weight of -LA and -LG, respectively, as illustrated in Figure 2.

FIGURE 2. Representative protein profile from WP-BRA supplements evaluated by SDSPAGE. -LA = -lactalbumin; -LG = -lactoglobulin; MW Stand = Molecular weight standard; WP-BRA = whey protein supplements produced by Brazilian companies.

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Almeida et al.

FIGURE 3. Content of -LA (A) and -LG (variant A + variant B) (B) of WP-USA and
WP-BRA supplements. -LA = -lactalbumin; -LG = -lactoglobulin; WP-USA = whey
protein supplements produced by U.S. companies; WP-BRA = whey protein supplements
produced by Brazilian companies. The symbol (p < 0.05) denotes significantly different
from WP-BRA.

The content of -LA and -LG of the WP-USA and WP-BRA supplements are
presented in the Figure 3. There was a difference (p < 0.05) in content of -LA and
-LG (variant A + variant B) between the WP-USA and WP-BRA supplements
(-LA: 5.38 2.0 vs. 3.34 2.2 mg/100 g; -LG: 7.25 4.1 vs. 5.43 2.3 mg/100 g,
for WP-USA and WP-BRA, respectively) (Figure 3). Figure 4 illustrates a representative chromatogram of -LA and -LG (variant A and variant B).
Amino Acids
The content of free EAA and free BCAA of the WP-USA and WP-BRA supplements are exhibited in the Table 1. In terms of free EAA, there was no difference (p
> 0.05) between WP-USA and WP-BRA (378.1 854.9 vs. 118.7 183.0 mg/100 g,
respectively). However, the content of free BCAA was greater (p < 0.05) on WPUSA than on WP-BRA (332.0 816.7 vs. 28.9 49.9 mg/100 g, respectively).

FIGURE 4. Representative chromatogram of one of the WP-USA supplements. lactalbumin (a, 26 min); -lactoglobulin variant A (b, 36 min); -lactoglobulin variant B
(c, 39 min).

Protein and Amino Acid Analysis in Supplements

TABLE 1. Concentration (mg/100 g) of free Essential Amino Acid (EAA) and the Free
Branch-chain Amino Acids (BCAA) of WP-USA and WP-BRA Supplements
Amino acid

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Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Valine
 EAA
 BCAA

WP-USA

WP-BRA

2.7 1.9
95.5 232.3
125.6 305.9
21.1 18.5
5.2 8.1
13.8 17.5
3.1 4.3
110.9 278.8
378.1 854.9
332.0 816.7

11.6 21.8
7.8 13.9
11.3 19.0
47.2 61.1
5.1 7.6
16.5 28.0
9.2 16.7
9.7 17.0
118.7 183.0
28.9 49.9

DISCUSSION
The present study was designed with the purpose of evaluating and comparing the
content of total protein (TP), -LA, -LG, free EAA, and free BCAA, among WP
supplements produced by U.S. companies and by Brazilian companies. Overall,
the result of the study demonstrated that the contents of TP, -LA, -LG, and free
BCAA were greater in WP-USA than in WP-BRA; however, the content of free
EAA was similar between WP-USA and WP-BRA. In addition, from the 10 WPUSA supplements evaluated, 40% exhibited their total protein content below the
value specified on the label; whereas 70% of the 10 WP-BRA supplements showed
the same discrepancy.
Tests carried out by Consumer Lab (2014), a private company specialized in the
assessment of food quality, also demonstrated similar results: from 24 commercially
produced WP by U.S. companies, 31% of products tests failed in their quality assurance. As reported by Proteste Organization (2014), agency for consumer protection
in Brazil, amongst the 28 WP-BRA brands evaluated, 53% exhibited lower values
in total protein content as compared to their respective labels.
Differences found in the contents of TP, -LA, -LG, and free BCAA between
WP-USA and WP-BRA supplements are potentially due to the technology utilized
to manufacture the supplements or in the protein composition of cow milk used
for obtaining whey, which can be influenced by the breed, lactation stage, or diet

(Murphy & OMara, 1993; Walstra et al., 2006). As described by Remond,

Coulon,
Nicloux, and Levieux (1999), a gradual and significant decrease in -LG and -LA
contents during the lactation period can be observed in different cow breeds. In a
study performed by Wu and Huber (1994), the supplementation of dairy cows with
high fat diets resulted in a decreased protein concentration in milk. Furthermore,

according to Jimenez,
Cuenca, Jurado, Corona, and Urista (2012), the chemical additives and other factors, such as pressure, temperature, agitation rate and holding
time, affected pH, protein conformation and the purity of WP supplements.
One another possible explanation for the differences found in the contents of
TP, -LA, -LG, and free BCAA between and among WP-USA and WP-BRA
supplements may be related to the whey protein type. In the present study of
the 10 WP-BRA supplements evaluated, 5 were WP isolate (WPI) and 5 WP

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Almeida et al.

concentrate (WPC). Of the 10 WP-USA supplements evaluated, 5 were WPI and

5 were WP concentrate and isolate (WPCI). Manufacturing WPC involves ultrafiltration of whey to concentrate proteins and diafiltration to remove most of lactose,
minerals and other low molecular weight components. The retentate is usually further concentrated by evaporation before spray drying to improve the physical properties of the powder, so that the finished dry product contains up to 80% of protein
(Carunchia, Croissant, & Drake, 2005). WPI is obtained by ion exchange, removing sufficient nonprotein constituents from whey so that the finished dry product
contains 90% or more protein (Urista et al., 2011). Therefore, WPC should contain
up to 80% of protein, WPI more than 90% of protein and WPCI protein concentration will depend on the amount used of each WPI and WPC, during its manufacture
(Mayyada & Howard, 2011). In our study of 10 WP-USA, five were WPI and five
were WPCI. Because all of the WP-USA supplements evaluated contained WPI,
a better protein and amino acid profile compared to the WP-BRA supplements
would be expected, since half of the WP-BRA products analyzed were WPC. It
is important to point out that although the TP contents found in some WP supplements evaluated in the present study were in accordance with those described
on the label, none of evaluated WP supplements achieved the contents of TP described in the literature (i.e. up to 80% for WPC and more than 90% for WPI
supplements).
Whey is one of two protein fractions of dairy protein, the other being casein.
Bovine protein is made up of approximately 7080% casein; whey protein makes
up the remaining 2030%. Whey is the water-soluble fraction that is extracted from
casein during the process of coagulation (Walstra et al., 2006). Casein is also a rich
source of EAA (Sindayikengera , & Wen-shui, 2006). In this study, a similarity
in the total content of free EAA between WP-USA and WP-BRA was observed.
Even with differences among the brands found through other parameters (TP, LA, and -LG), this similarity can refer an apparent failure in the extraction of
whey proteins with a possible presence of casein, or through the addition from other
sources of protein.
Some manufacturers add specific amino acids, such as BCAA, in their formulations in order to fortify protein supplements, as an effort to nullify the negative
effects during processing (Judy, & Ira, 2001). This was observed in some brands
of WP-USA evaluated, which explains the greater values found for valine, leucine,
and isoleucine in WP-USA. The BCAA (leucine, isoleucine, and valine) have been
investigated for their anticatabolic and anabolic effects (especially leucine) (Hulmi,
Christopher, & Jeffrey, 2010). In skeletal muscle in vitro, increasing the concentration of the three BCAA or l-leucine alone reproduces the effects of increasing
the supply of all amino acids in stimulating protein synthesis and inhibiting protein
degradation (Miller, Tipton, Chinkes, Wolf, & Wolfe, 2003). Thus, within the context of the potential benefits associated with protein intakes, it is also important to
consider the branched chain amino acid (Borsheim, Tipton, Wolf, & Wolfe, 2002;
Maughan, 2013). Miller et al. (2003) proposed the existence of a dose-response
relation between muscle protein synthesis and amino acid consumption after resistance exercise. The authors documented a post-exercise stimulation of muscle
protein synthesis almost twice as great after ingestion of 6 g compared with only
3 g of EAAs.

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Protein and Amino Acid Analysis in Supplements

Physical exercise, in general, requires a higher protein intake, which is due to an

increased use of amino acids as an energy source in metabolism (Ha & Zemel, 2003;
Layman, 2003); however, the variation of amino acid composition in whey protein
supplements may influence the intensity of the response expected. Therefore, the
nutritional value of a dietary protein should be taken into account, particularly the
BCAA, responsible for stimulate muscle protein synthesis (Millward, Layman, &
Schaafsma, 2008).
Consumers expect that a supplement indeed contains the ingredients and
amounts as listed on the label; however, this is not always the case and evidence
for poor quality control has been frequently reported (FDA, 2014). It is difficult
to assess the extent of these problems, because there is no comprehensive testing program for these supplements to ensure the composition and effects of these
products; meaning that the manufacturer is responsible for ensuring that the Supplement Facts label and ingredient list are accurate, that the dietary ingredients
are safe, and that the content matches the amount declared on the label (Kreider
et al., 2010).
CONCLUSIONS
Overall, the WP supplements analyzed had variable amounts of TP, -LA, -LG,
free EAA, and free BCAA. The results of the present study indicated that the WPUSA supplements showed better nutritional quality, as evaluated through contents
of TP, -LA, -LG, and free BCAA than WP-BRA brands. This data emphasize
the necessity to improvement the oversight with respect to the quality of WP supplements sold on the market, regardless of the origin of the manufacture. Therefore, periodic evaluation of products and verification of labeling are advisable in
order to guarantee product quality for consumers.
ACKNOWLEDGMENTS
The authors would thank Ricky Toledano for preparing the English version of the
manuscript and The Research Foundation of the State of Rio de JaneiroFAPERJ
and National Council of Technological and Scientific Development (CNPq) by
provided financial support for the study (process no. E-26/103.003/2012 and
311361/2013-7, respectively). Cristine C. Almeida and Marion P. Costa were supported by scholarship from CNPq.
Declaration of interest: The authors have no conflicts of interest that are directly
relevant to the content of this manuscript. The authors alone are responsible for the
content and writing of the article.
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