Journal of Food and Nutrition Research (ISSN 1336-8672)

Vol. 56, 2017, No. 2, pp. 101–108

Evaluation of antioxidant activity of whey protein to improve

cholesterol oxidation stability in fresh white cheese from buttermilk
Paulina Bierzuńska – Dorota Cais-Sokolińska –
Magdalena Rudzińska – Anna Gramza-Michałowska

Summary
The health-promoting value of fresh white cheese made from buttermilk can be increased by adding to it a whey protein
concentrate (WPC). At the same time, the antioxidant potential of whey protein can be used to stabilize the lipid frac-
tion. WPC-enriched cheese had higher ferric reducing antioxidant power (FRAP) values than the control buttermilk
cheese. The 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity (DPPH) was also higher in WPC-enriched cheese,
but not as noticeable as in the case of the FRAP assay results. A strong correlation was established between FRAP and
DPPH antioxidant activity values, and the amount of cholesterol oxidation products in WPC-enriched cheese. The
longer the WPC-enriched buttermilk cheese was stored, the lower the number of cholesterol oxidation products (mostly
7β-hydroxycholesterol and 5,6-β-epoxycholesterol) was determined. After 30 days, the content of total cholesterol oxi-
dation products in WPC-enriched cheese was 4.7-fold lower than in the control cheese.

Keywords
cheese; buttermilk; antioxidant activity; whey protein

Dairy industry is one of the most innovative polyunsaturated fatty acids, buttermilk is suscepti-
food sectors. Consumers seek out those dairy ble to oxidative deterioration [4]. MFGM contains
products that provide for the proper physiological a mixture of proteins, glycoproteins and phospho­
functioning of the body and that contain bioac- lipids, all of which can act as emulsifiers and help
tive components [1, 2]. Products with new or im- the fat globule remain suspended in milk. Since
proved ingredients and health-promoting proper- buttermilk contains MFGM (along with casein),
ties are appearing on the market, including acid it is an interesting source of ingredients with the
whey cheeses. The most important cheese in this potential to impart certain specific characteristics
assortment group is fresh white cheese (tvorog, to cheese made from it [5]. Using buttermilk in
quark, twaróg, biały ser, farmer cheese, Weißkäse, cheese production is also justified economically.
beli  sir, tvaroh). The raw substance used in the In traditional cheese-making, casein is almost
production of traditional fresh white cheese is the exclusive curd structure. Casein contains four
milk subjected to acid or acid–rennet coagulation. fractions: αS1-casein, αs2-casein, β-casein, and
Milk can also be replaced by buttermilk or a mix- κ-casein. Almost the entire whey protein frac-
ture of non-fermented milk and BM. The use of tion, β-lactoglobulin (β-LG) and α-lactoalbumin
buttermilk in cheese was mentioned by Ferreiro (α-LA), upon precipitating at isoelectric point
et al. [3]. Buttermilk resembles skim milk in com- and curd processing, remains in solution and is
position and appearance, but contains most of the lost in whey. This represents a very significant
milk fat globule membrane (MFGM), which is huge loss, because whey proteins have antioxi-
composed of phospholipids, including phosphati- dant properties [6]. These properties depend on,
dylcholine (lecithin), phosphatidylethanolamine among others, the presence of sulfuric amino
and sphingomyelin. Due to high concentration of acids in the structure. More precisely, β-LG is the

Paulina Bierzuńska, Dorota Cais-Sokolińska, Department of Dairy Technology, Faculty of Food Science and Nutrition,
Poznań University of Life Sciences, ul. Wojska Polskiego 31, 60-624 Poznań, Poland.
Magdalena Rudzińska, Department of Food Chemistry and Instrumental Analysis, Faculty of Food Science and Nutrition,
Poznań University of Life Sciences, ul. Wojska Polskiego 31, 60-624 Poznań, Poland.
Anna Gramza-Michałowska, Department of Food Service and Catering, Faculty of Food Science and Nutrition,
Poznań University of Life Sciences, ul. Wojska Polskiego 31, 60-624 Poznań, Poland.
Correspondence author:
Dorota Cais-Sokolińska, tel. +48618487317, e-mail: cais@up.poznan.pl

© 2017 National Agricultural and Food Centre (Slovakia) 101

Bierzuńska, P. et al. J. Food Nutr. Res., Vol. 56, 2017, pp. 101–108

source of γ-glutamylcysteine dipeptide, the pre- powder product, sweet WPC80 (Spomlek Dairy
cursor of glutathione that possesses strong anti- Cooperative, Radzyń Podlaski, Poland), was dis-
oxidant properties [7]. The main components of solved in 5 l of whey after the cheese had been
antioxidant peptides are the amino acid residues made from buttermilk. Then, a solution of 4 cm3
of histidine, tyrosine, methionine, lysine, and tryp- of 60% CaCl2 was added to a 50-litre mixture of
tophan, which are also antioxidants in their free buttermilk and WPC to integrate the buttermilk-
form. It is not only the amino acid sequence, but based casein and WPC-enriched whey proteins
also the spatial configuration of the peptide, that introduced to it. From that point on, the cheese-
give it its antioxidant properties [8, 9]. The ability making process proceeded unchanged. The tech-
to neutralize free radicals and inhibit enzymatic nical and technological conditions of the butter-
and non-enzymatic oxidation of lipids is exhibit- milk processing, with and without the addition of
ed by a fragment of β-LG, a peptide with the se- WPC, were chosen so that coagulation occurred
quence WYSLAMAASDI. This peptide, obtained after 2 h at 50–52 °C. The curd had titratable acid-
through proteolysis by corolase PP, demonstrated ity of 0.7  % (expressed as percentage of lactic
a greater ability to neutralize free radicals than did acid) and pH of 4.6. It was sliced into cubes 10 cm
butylhydroxyanisole. β-LG and α-LA may also re- long, and then 4 cm long, which were later stirred
lease peptides of sequences MHIRL and YVEEL, and heated to 34 °C. The heating rate was 1 °C per
both exhibiting antioxidant properties [10]. 10  min. The titratable acidity of the whey after
In dairy products, the initiators of free-radical heating did not exceed 0.6 % of lactic acid. Drain-
processes are the cholesterol oxidation products ing was performed in a layer (h = 10 cm) on a spe-
(COPs), which are highly reactive components. cial curd-finishing table. Ironing took 3.5 h at the
The most bioactive and cytotoxic COPs include initial and final temperatures of 20 °C and 10 °C,
cholesterol 25-hydroxylase (25-OHC), cholesta- respectively, and at a maximum pressing force of
netriol (triolC), 7α- and 7β-hydroxycholesterol 3 kg per 1 kg of cheese. Fresh white cheese made
(7α-OHC and 7β-OHC), 7-ketocholesterol from buttermilk and buttermilk with WPC was
(7-ketoC) and epoxy derivatives of cholesterol, then shaped into cubes weighing 170 g each and
5.6β- and 5.6α-epoxycholesterol (β-epoxyC and packaged in paper foil. The cheese was stored
α-epoxyC) [11]. Their presence in milk depends at 4  ± 0.5 °C and examined within the first 24 h
on the initial content of cholesterol, tempera- (Day 0) and then after 10, 20 and 30 days.
ture and time of storage, as well as on availability
of light and oxygen [11, 12]. Milk fat in cheeses Compositional analysis
is considered oxidatively more stable than, for Moisture in the cheese was determined by
example, milk powder. Only limited data are avail- standard methods AOAC 926.08 [13]. The con-
able in scientific literature on analysis of oxysterols tent of total nitrogen (TN) was determined by the
in fresh cheese, and even fewer studies regarded Kjeldahl method [14] with the assistance of the
oxysterols in buttermilk. distillation unit of the Kjeltec System 1026 appara-
The objective of this study was to utilize whey tus (Tecator, Örebro, Sweden). Content of casein
proteins and employ their antioxidant potential nitrogen (CN), non-casein nitrogen (NCN) and
to stabilize the lipid fraction of buttermilk cheese. non-protein nitrogen (NPN) were determined ac-
The evaluation of stability was based on the de- cording to Svanborg et al. [15]. Content of total
termination of cholesterol oxidation products in protein (TP) and whey protein (WP) were calcu-
cheeses made from buttermilk. lated according to the following equations:
𝑇𝑇𝑇𝑇 = (𝑇𝑇𝑇𝑇 − 𝑁𝑁𝑁𝑁𝑁𝑁) × 6.38 (1)
Materials and methods 𝑊𝑊𝑊𝑊 = (𝑁𝑁𝑁𝑁𝑁𝑁 − 𝑁𝑁𝑁𝑁𝑁𝑁) × 6.38 (2)
Collection, packaging, storage and sampling of where number 6.38 represents the factor indicated
cheeses for protein derived from milk.
The research material was fresh white cheese Fat content and titratable acidity (expressed as
made from buttermilk, a left-over after the in- percentage of lactic acid) in the cheese were deter-
dustrial production of butter from cream (Great mined by standard methods ISO 1735 and AOAC
Poland). Model cheese parts (n  = 7) were made 920.124, respectively [16, 17]. The pH value of the
on a  pilot scale (Poznań/Września, Poland). The examined cheeses was determined by standard
cheese was prepared from buttermilk, with or methods using a pH-meter CP-315 (Elmetron,
without the addition of whey protein concentrate Zabrze, Poland), which was equipped with a com-
(WPC). For this purpose, 2.2 kg of a commercial bined ESAgP-301W electrode (Eurosensor, Gli-

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 Antioxidant activity of whey protein to improve cholesterol oxidation stability in cheese from buttermilk

wice, Poland), consisting of a glass half-cell and metry utilizing our library and published data [21].
a saturated chloro-silver half-cell [18].
Fat from milk and cheese was extracted Ferric reducing antioxidant power assay
according to Röse–Gottlieb procedure following Ferric reducing antioxidant power (FRAP)
the method ISO 14156 [19]. evaluates the ability of the analysed substance
to reduce the complex of Fe(III)–2,4,6-tris(2-
Determination of cholesterol pirydyl)-s-triazine (TPTZ) to the form of Fe(II)–
The cholesterol content was determined using TPTZ [23]. The intensity of the blue colour,
AOCS Official Methods Ch. 6–91 [20] by gas chro- measured spectrophotometrically at 583 nm using
matography, after saponification with 1 mol·l-1 apparatus RayLeigh UV-1601 (Beijing Rayleigh
KOH in methanol for 18 h at room temperature Analytical Instruments, Beijing, China) was
and triple extraction of the unsaponifiables with linearly correlated with the reducing agent con-
hexane : tert-butyl methyl ether (MTBE) 1 : 1,  v/v. centration. Results were presented as millimoles
The solvent was evaporated under a stream of ni- of Fe2+ per litre, based on a standard curve
trogen. Dry residues were dissolved in 0.1 ml pyri-
𝑦𝑦 = 0.0001𝑥𝑥 + 0.0113; 𝑟𝑟 2 = 0.9938 (3)
dine and silylated with 0.4 ml of Sylon BTZ (Su-
pelco, Bellefonte, Pennsylvania, USA). Derivatives where y is absorbance, x is standard (Fe II) or
of the sterols were separated on a gas chromato- evaluated sample concentration and r2 is coeffi-
graph HP 6890 equipped with a DB-35MS capil- cient of determination.
lary column (25 m × 0.20 mm; with a  stationary
phase film of 0.33  mm; J&W Scientific, Folsom, DPPH radical-scavenging assay
California, USA). A sample was injected in split- The ability of the antioxidant to scavenge
less mode. Column temperature was held at 100 °C stable 1,1-diphenyl-2-picrylhydrazyl (DPPH)
for 5 min, then programmed to rise to 250 °C at radicals was evaluated spectrophotometrically at
25 °C∙min-1, held for 1 min, then to rise to 290 °C 517  nm using RayLeigh UV-1601, in relation to
at 3 °C∙min-1, and held for 20 min. The detector the radical-scavenging ability of the reference sub-
temperature was set to 300 °C. Hydrogen was used stance, 6-hydroxy-2,5,7,8-tetramethylchromane-
as a carrier gas at a flow rate of 1.5 ml∙min-1. 5a- 2-car­boxylic acid (Trolox) [24]. The absorbance
Cholestane was used as the internal standard for decrease is a result of the radical-scavenging abil-
sterol quantification. Identification was based on ity of the substance. Results were presented as
the retention data for compounds previously veri- milli­moles of Trolox equivalents (TE) per kilo-
fied by mass spectrometry [21]. gram, based on a standard curve:
𝑦𝑦 = 83.8𝑥𝑥; 𝑟𝑟 2 = 0.9635 (4)
Determination of cholesterol oxidation products
COPs were determined according to the where y is percentage of reduced DPPH radical,
methodology described by Przygoński et al. x is standard (Trolox) or evaluated sample concen-
[22]. Isolated fats were esterified with 10% so- tration and r2 is coefficient of determination.
dium methylate in MTBE (4 : 6, v/v) and choles-
terol together with its oxidation products, were Statistical analysis
extracted with chloroform and then fractionated The obtained data (n = 7) were expressed as
on a  SEP–PAK NH2 column (Waters, Milford, mean values and the respective standard devia-
Connecticut, USA). Chromatographic analysis of tions, and analysed by using repeated-measures
silylated oxysterols was performed on a Hewlett- ANOVA. The correlation coefficient significance
Packard 6890 apparatus (Hewlett-Packard, tests were based on the assumption of normal dis-
Palo Alto, California, USA). Separation was run tribution of the residual value of y variable and
on a DB-5 capillary column (30  m  × 0.25 mm, an  equal residual value variation for all values of
with a stationary phase film of 0.25 µm; J&W the x variable. In order to eliminate deviations
Scientific), using a programmed temperature at from Pearson’s distribution linearity, which could
a rate of 25 °C∙min-1 from 60 °C to 270 °C, fol- cause an increase of the square sum of deviations
lowed by 2.5 °C∙min-1 until 290 °C was reached. from the regression line, a scatter diagram analy-
Helium was used as a carrier gas at a flow rate sis was performed. Paired t-tests were used for the
of 1  cm3∙min-1. The injector and detector were calculations. For the verification of statistical hy-
held at 300 °C and the split ratio at 1 : 40. 19-Hy- potheses, a level of significance of α = 0.05 was
droxycholesterol was used as an internal standard. adopted. The statistical calculations were made
Oxysterols were identified using retention data for using Statistica data analysis software system, ver-
compounds previously verified by mass spectro­ sion 10 (StatSoft, Tulsa, Oklahoma, USA).

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Results and discussion adds flavour and it provides certain functional and
technological properties due to the high phospho-
The buttermilk used to produce the cheese lipid content [31]. Buttermilk is higher in MFGM
contained 0.4 % fat, 3.3 % protein and 3.8 % lac- protein and phospholipids than skim milk, which
tose. Titratable acidity was at the level of 0.4  %. may alter the functional properties of its WPC
WPC80 contained 78.8  % protein, 1.6  % lactose [14]. In the dairy industry, various techniques were
and 1.2  % mineral salts. Titratable acidity, pH, developed for whey protein introduction [26].
moisture, fat and fat in dry matter did not change Most of these are based on adding denatured and
significantly with WPC usage in fresh white cheese aggregated whey proteins to cheese milk. What
made from buttermilk with and without WPC. may be problematic is to determine the size and
An important result of the study was to de­ water-holding capacity of such aggregated whey
monstrate that it was possible to incorporate whey proteins [32]. Larger aggregates readily interfere
proteins into buttermilk cheese (ChB). The inclu- with the casein network and are poorly retained
sion of WPC and CaCl2 (ChB-WPC) significantly in cheese curd. According to Saffon et al. [32],
increased the total content of protein in the cheese another issue associated with the incorporation
by 2.1  %, p < 0.05 (total protein content in the of aggregated proteins is related to their effect
ChB-WPC was 172.6 g∙kg-1) (Tab. 1). The ratio of on cheese moisture. Excessive cheese moisture is
casein to whey protein in ChB was only 20.3, while a common defect that limits the amount of whey
the ratio was 17.3 (p < 0.05) in ChB-WPC. Part of protein that can be added to cheese milk. Whey
the whey proteins integrated in casein could have proteins have the ability to bind, particularly
come from concentrate and the buttermilk itself. with serum-phase κ-casein after the dissociation
The literature repeatedly emphasized the nutri- of κ-casein from micelles [33]. Ye et al. demon-
tional and structure-forming role of whey proteins strated that β-LG and α-LA associated with the
[25–27] and the properties of buttermilk cheese MFGM by thiol–disulfide bonds in heated milk
[28–30]. After being long considered a  low-value [34, 35]. Co-denaturation of cheese whey and
by-product, buttermilk is now widely used both as butter­milk concentrates leads to the formation
the basic ingredient in beverages and certain types of protein aggregates. Higher proportions of but-
of cheese, and as an ingredient of other foods. It termilk protein in the mixtures increased yields
is characterized by a high emulsifying capacity, it and decreased the water-holding capacities of the

Tab. 1. Physico-chemical characteristics and antioxidant activity of fresh white cheese made from buttermilk.
Parameters Cheese without WPC Cheese with WPC
Moisture [g∙kg-1] 782.5 ± 1.3 a 785.6 ± 1.6 a
Total nitrogen [g∙kg-1] 28.6 ± 0.3 a 30.3 ± 0.4 b
Non-protein nitrogen [g∙kg-1] 3.1 ± 0.7 a 3.2 ± 0.5 a
Non-casein nitrogen [g∙kg-1] 0.2 ± 0.7 a 4.5 ± 0.9 a
Total protein [g∙kg-1] 169.1 ± 0.5 a 172.6 ± 0.3 b
Casein [g∙kg-1] 142.3 ± 0.1 a 143.3 ± 0.4 a
Whey protein [g∙kg-1] 7.0 ± 0.4 a 8.3 ± 0.5 b
Total protein in dry matter [%] 77.8 a 80.5 b
Fat [g∙kg-1] 21.2 ± 0.5 a 20.8 ± 1.0 a
Fat in dry matter [%] 9.8 a 9.7 a
Titratable acidity [%] 1.6 ± 0.1 a 1.6 ± 0.1 a
pH 4.62 ± 0.02 a 4.60 ± 0.01 a
Salt [g∙kg-1] 0.8 ± 0.4 a 1.1 ± 0.3 a
Cholesterol [g∙kg-1] 12.32 ± 1.85 a 12.21 ± 1.32 a
FRAP [mmol∙l-1] 0.223 ± 0.010 a 0.687 ± 0.007 b
DPPH [mmol∙kg-1] 0.274 ± 0.042 a 0.334 ± 0.032 b
Values represent mean ± standard deviation (n = 7). Different small letters in superscript in a row indicate statistically significant
differences at the level a = 0.05.
WPC – whey protein concentrate. Titratable acidity is expressed as percentage of lactic acid. FRAP – ferric reducing antioxidant
power expressed as millimoles of Fe2+; DPPH – antiradical power expressed as millimoles of Trolox equivalents.

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Tab. 2. Antioxidant activity of fresh white cheese made from buttermilk during storage.
Cheese without WPC Cheese with WPC
Storage [d]
FRAP [mmol∙l-1] DPPH [mmol∙kg-1] FRAP [mmol∙l-1] DPPH [mmol∙kg-1]
0 0.223 ± 0.010 b 0.274 ± 0.042 b 0.687 ± 0.007 d 0.334 ± 0.032 c
10 0.239 ± 0.022 b 0.216 ± 0.009 a 0.689 ± 0.013 d 0.290 ± 0.020 b
20 0.189 ± 0.019 a 0.209 ± 0.028 a 0.414 ± 0.029 c 0.285 ± 0.015 b
30 0.206 ± 0.036 b 0.186 ± 0.015 a 0.257 ± 0.017 b 0.269 ± 0.058 b
Values represent mean ± standard deviation (n = 7). Different small letters in superscript in columns indicate statistically sig-
nificant differences at the level a = 0.05.
WPC – whey protein concentrate. FRAP – ferric reducing antioxidant power expressed as millimoles of Fe2+; DPPH – antiradical
power expressed as millimoles of Trolox equivalents.

aggregates. Preventing the formation of disulfide α-epoxyC, than the buttermilk cheese (Day 0)
bonds between denatured whey proteins increased (Tab. 3). When ChB-WPC cheese was stored
WHC of such aggregates [32]. longer, the amount of oxysterols decreased by
The cholesterol content in ChB and ChB-WPC 80  %. After 30  days of storage, only 7β-OHC
cheese did not differ (12.32 g∙kg-1 and 12.21 g∙kg-1 and β-epoxyC were found in ChB-WPC cheese
fat, p > 0.05). This was the condition for studying at a  total level of 1.02 mg∙kg-1 (fat basis). ChB
the effect of the addition of WPC on its oxida- butter­milk cheese, meanwhile, after the same
tion. After considering the fat content in ChB and period of time, contained the following: 7α-OHC,
ChB-WPC cheese, average cholesterol content 7β-OHC, β-epoxyC, and α-epoxyC at a total level
was 258 mg∙kg-1. Cholesterol content was much of 4.88 mg∙kg-1 (fat basis). On Day 0 and Day 10,
lower than indicated by Fletouris et al. [36] in WPC-free cheese contained 25-OHC and 7-ketoC.
mozzarella (714 mg∙kg-1), gouda (881 mg∙kg-1), A strong correlation was observed between the
feta (681 mg∙kg-1) or butter (2281 mg∙kg-1). Cho- content of oxysterols in ChB-WPC cheese and
lesterol levels in dairy products depend, among FRAP (r = 0.793) and DPPH (r = 0.990). ChB
other factors, on fat content, heat treatment, milk cheese without whey protein had much smaller
homogenization and type of lactic acid bacteria. correlation coefficients of 0.215 and 0.789 for
Milk contains approximately 120 mg of cholesterol FRAP and DPPH, respectively. These coefficient
per kilogram [36]. According to Kovacs et al. [37], values indicated that the quantity of oxysterols
low-fat dairy products have a larger share of cho- could not be compared with the size of the simul-
lesterol in fat than high-fat ones, as the small fatty taneous antioxidant activity of whey proteins. The
beads, which accumulate cholesterol, have a rela- samples with WPC addition were characterized
tively large surface area. by higher FRAP values than the control cheese
On Day 0 and Day 10 of storage, the cheese made from buttermilk. This higher activity was
with WPC showed a three-fold higher (p  < 0.05) probably due to the presence of peptides in WPC.
antioxidant activity (ΔFRAP = 0.464 mmol∙l-1 DPPH radical-scavenging activity was also higher
and ΔFRAP = 0.450 mmol∙l-1, respectively) than in WPC-enriched cheese, but not that markedly as
ChB cheese (Tab. 1, Tab. 2). After 30 days, FRAP in the case of the FRAP assay results. Thus, the re-
of WPC cheese decreased by 62.6  %, but only sults showed a significant increase of antioxidant
by 7.6  % in the case of the cheese made from potential of samples with WPC addition over the
butter­milk. However, after the same time, their 30 days of storage (Tab. 2). The expected antioxi-
anti­oxidant activities did not differ significantly dant effect occured only in the subsequent period
(0.206  mmol∙l-1 and 0.257 mmol∙l-1, respectively). of storage. It seems that the method involving ad-
After production and during the entire storage dition of CaCl2 was effective in stabilizing cho-
time, WPC cheese had a higher antiradical power lesterol against oxidation. The addition of whey
than the cheese made solely from buttermilk. protein concentrate was also justified, although
After storage, antiradical power was decreased in a better solution might be to introduce serum pro-
WPC cheese (ΔDPPH = 32.1 %) as well as in ChB tein concentrate (SPC). SPC obtained by micro-
(ΔDPPH = 19.5 %). filtration from raw milk was referred to as native
Antioxidant activity of whey proteins had a sig- whey protein, milk microfiltrate protein, virgin
nificant impact on the resistance of chole­sterol whey protein or milk serum protein [38, 39]. Com-
to oxidation. WPC cheese contained 4.5-fold parison of 80% WPC and SPC with commercial
(p < 0.05) more oxysterols, mainly 7α-OHC and products showed higher levels of lipid oxidation in

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Tab. 3. Levels of oxysterols in fresh white cheese made from buttermilk.

7α-OHC 7β-OHC β-epoxyC α-epoxyC triolC 25-OHC 7-ketoC Σ COPs
Storage [d]
[mg·kg-1] [mg·kg-1] [mg·kg-1] [mg·kg-1] [mg·kg-1] [mg·kg-1] [mg·kg-1] [mg·kg-1]
Cheese without WPC
0 0.12 ± 0.01 a 0.29 ± 0.01 b 0.28 ± 0.01 a 0.04 ± 0.01 a nd 0.35 ± 0.02 a 0.09 ± 0.03 a 1.17
10 0.13 ± 0.02 a 0.10 ± 0.01 a 1.18 ± 0.04 c 1.56 ± 0.11 d nd 0.37 ± 0.01 a 0.91 ± 0.01 c 4.25
20 nd 1.28 ± 0.02 d nd 0.72 ± 0.07 b nd nd nd 2.00
30 0.92 ± 0.07 b 1.25 ± 0.08 d 1.28 ± 0.04 c 1.43 ± 0.01 d nd nd nd 4.88
Cheese with WPC
0 1.21 ± 0.07 c 0.74 ± 0.08 c 0.90 ± 0.04 c 1.01 ± 0.01 c nd 0.76 ± 0.01 b 0.53 ± 0.01 b 5.15
10 nd 1.90 ± 0.02 e 0.59 ± 0.01 b nd nd nd nd 2.49
20 nd 1.11 ± 0.08 d nd 0.47 ± 0.03 b nd nd nd 1.58
30 nd 0.62 ± 0.05 c 0.40 ± 0.05 a nd nd nd nd 1.02
Values represent mean ± standard deviation (n = 7). Different small letters in superscript in columns indicate statistically sig-
nificant differences at the level a = 0.05.
WPC – whey protein concentrate. 7α-OHC – 7α-hydroxycholesterol; 7β-OHC – 7β-hydroxycholesterol; β-epoxyC –
5.6β-epoxycholesterol; α-epoxyC – 5.6α-epoxycholesterol; triolC – cholestanetriol; 25-OHC – 25-hydroxycholesterol; 7-ketoC –
7-ketocholesterol; Σ COPs – sum of cholesterol oxidation products. nd – not detected.

commercial WPC products [40]. SPC has unique which persisted during 30 days of storage. The ad-
functionalities, such as excellent solubility, gelling dition of whey protein to cheese reduced the for-
after heat treatment and foaming properties [41]. mation of 7α-OHC, 25-OHC and 7-ketoC during
Cholesterol undergoes autoxidation in contact storage. The formation of β-epoxyC and α-epoxyC
with air, the degree of formation of cholesterol was reduced in the buttermilk cheese with added
oxidation products being mainly dependent on whey protein. At the end of the storage period, to-
storage conditions [12]. Sander et al. [42] showed tal content of cholesterol oxidation products was
that cheese spread, cottage cheese, evaporated lower than in the control cheese. No triolC, which
milk, and whole milk did not contain any of is the most toxic form of oxycholesterol, was found
the COPs. In cream cheese, only α-epoxyC and in the cheeses. This cheese, enriched with whey
7-ketoC were detected at 9 mg∙kg-1 and 3 mg∙kg-1, protein, can broaden the range of innovative and
respectively. In cheese spread, COPs were detect- health-promoting dairy products.
ed at 7 mg∙kg-1 (fat basis) [43]. According to those
authors, the probability of COPs being formed in
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