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Microparticulation of whey protein: related factors affecting the solubility

Article  in  Zeitschrift für Lebensmittel-Untersuchung und -Forschung · October 1994

DOI: 10.1007/BF01193314

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Z LebensmUnters Forsch (1994) 199:289-293 Zeitschrift fL~r

@ Springer-Verlag 1994

Original paper
Microparticulation of whey protein:
related factors affecting the solubility
B~irbel Lieske, Gerd Konrad
FachhochschuleAnhalt, FachbereichLandwirtschaft,()kotrophologieund Landespflege,Augenstelle Oranienburg, SachsenhausenerStrasse 7,
D-16515 Oranienburg, Germany

Received December29, 1993; revised version April 7, 1994

Mikropartikulierung yon Molkenprotein: Introduction

Faktoren, die die Liislichkeit beeinflussen
Simplesse 100 is the only protein-based fat substitute
Zusammenfassung. Simplesse 100, Fettsubstitut auf Mol- obtained from whey protein as a result of denaturing
kenbasis, zeigt ein gutes L6sungsverhalten. Da die Entste- treatments, like thermodenaturation under moderate acidic
hung von Mikropartikeln mit thermischen Veffahrens- conditions, followed by thermocoagulation and microparti-
schritten verbunden ist, ist ein solches Verhalten unge- culation [1, 2]. The sizes in whey protein particles produced
w6hnlich. Zur Klirung dieses Phinomens wurden gelchro- in this way are comparable with those of homogenized milk
matographische Trennungen an Sephadex G-100 bzw. an fat globules. In the narrow range between 0.5 gm and
Sephacryl S-1000 sowie die SDS-PAGE herangezogen, 3.0 gm particle size, Simplesse 100 is perceived orally to
untersttitzt durch UV-Studien, analytische Techniken und be like high-quality fats, as well as exhibiting their
Zentrifugation bei 23.000 g. Die Ergebnisse lassen den structural characteristics. Thus, this feature represents a
Schlu6 zu, dab das L6sungsverhalten mikropartikulierten new kind of function of whey protein.
Molkenproteins im wesentlichen yon zwei Effekten be- Considering that Simplesse 100 is made from relatively
stimmt wird, (1) der optimalen Entfaltung der Proteinmo- crude whey protein concentrate of about 50% protein in the
lektile und (2) der Stabilisierung der entfalteten Proteinmo- dry matter and that this protein has gone through several
lektile durch Kohlenhydrate. Beide Effekte favorisieren denaturing thermal steps, it still retains rather good solubi-
nicht-kovalente Bindungsmechanismen im mikropartiku- lity and digestibility [3]. This can be put down to the fact
lierten Protein und sind verantwortlich ftir physiko-funk- that: (1) whey protein remains soluble after thermal
tionelle und nutritive Eigenschaften. denaturation under acidic conditions [4-6] because any
disulphide interaction is excluded, and (2) lactose seems to
Abstract. Solubility of Simplesse 100, the only whey- be important for the thermal stability of microparticulated
based fat substitute, was found to be good, considering whey protein, as described previously [7] for various sugars
the fact that technology for preparation of Simplesse 100 is in connection with thermal denaturation and coagulation.
a sequence of thermal steps. To characterize this phenomen, In this work both factors were elucidated to find out
gel chromatography on Sephadex G-100, Sephacryl S-1000 inter-relations between solubility characteristics and corre-
and SDS-PAGE were used, supported by high-speed sepa- sponding structural orientation as prerequisites for micro-
ration, UV studies and analytical procedures. Results show particulation.
that the unusual solubility characteristic of microparticu-
lated whey protein is related to two molecular effects: (1)
optimal defolding of protein molecules and (2) stabilization
Materials and methods
of the defolded status by carbohydrate. Both effects were
considered to favour non-covalent bonds, which contribute
Whey products. Simplesse 100 dry, a microparticulated whey protein
to the outstanding physico-functional and nutritive proper- powder containing 50.4% protein and 27.1% lactose, was obtained
ties of microparticles. from Nutrasweet, Germany. Lacprodan 80, containing 78.0% protein
and 4.0% lactose, is an ultrafilteredwhey protein powder produced by
Danmark Protein. Raw milk whey was obtained by isoelectric
precipitation of casein at pH 4.6 using 1 N HC1. The coagulate was
separated at 6000 g for 10 rain. The acidic modification of raw milk
whey was carried out at 90~ for 10 rain at pH 3.8. After thermal
treatment the modified whey was rapidly cooled down to room
Correspondence to: B. Lieske temperature and neutralized to pH 6.4 with 1 N NaOH.
290

Table 1. The nitrogen solubility index (NSI) of different preparations

of whey protein at various pH (data in %) A28o
pH NSI of whey protein preparation:
1 ,ooo.
Lacprodan 80 Lacprodan 80 Simplesse 100
pH 6.5/90~ min dry
2.0 97.6 47.8 76.6 /)i
3.0 89.9 43.1 55.5
4.0 81.3 31.5 53.5 0,500.
5.0 80.5 32.1 51.8
6.0 92.1 55.2 57.5
7.0 94.5 53.4 58.3
8.0 94.8 55.4 66.2
,.,.....J
m
In the third column Lacprodan 80 has been previously heat treated at
pH 6.5 at 90~ for 5 rain a
5O 150 rnl

Determination of solubility. The nitrogen solubility index (NSI) was A280

examined by the centrifngation method using 2.5% protein solutions
and determination of the nitrogen content in the supernatant [8].
Turbidimetric measurements of the absorbance index (AI) were 1,000
carried out at 900 nm [9] using the Novaspec photometer (Pharmacia).

Chromatographicfractionations. Gel permeation chromatography was

performed using Sephadex G-t00 in a glass column (100 • 2.5 cm) and
!
0.02 mol/1 sodium citrate at pH 6.0 containing 0.02% sodium azide as 0,500
a preservative. The effluents were continuously monitored by measu-
ring the absorbance at 280 nm and collected in 3.0-ml fractions at a
flow rate of 35 ml/h. Gel permeation chromatography on Sephacryl S-
1000 was performed in a glass column (50 x 2.5 cm). The elution
buffer contained 3 mol/1 urea, 20 mmol/1 imidazole and 50 mmol/1
NaC1 and was adjusted to pH 6.9 with 1 N HC1. The effluents were b 50 150 ml:
collected in 3.0-ml fractions at a flow rate of 60 ml/h. The absorbances
of all fractions were measured at 280 nm. Fractions were further Fig. 1.a Elution profiles of Simplesse 100 (solid line) and Lacprodan
investigated by chemical methods and SDS-PAGE. 80 (dotted line) on a Sephacryl S-1000 column (0.2 mol/1 NaC1,
pH 6.9). b Elution profiles of Simplesse 100 and Lacprodan 80 on a
Chemical methods. Protein was determined by the Bio-Rad protein Sephacryl S-1000 column (3 mol/1 urea, 20 mmol/1 imidazole,
assay, whereby calibrations were made using solutions of 0.05%, 0.1% 50 mmol/1 NaC1, pH 6.9). The absorbance of all fractions were
and 0,2% Simplesse 100 or Lacprodan 80 prepared in elution buffer for measured at 280 nm (A280)
chromatography. As a microassay for lactose, a modified procedure of
the phenol-sulphuric acid method was used [10].
Nevertheless, microparticulated whey protein still has
Polyacrylamide gel electrophoresis. SDS-PAGE using tricin, according enough molecular flexibility to exhibit quite reasonable
to Schhgger and Jagow [11], was done in the Multiphor II Electro- physico-functionality and digestibility [3]. To elucidate the
phoresis System (Pharmacia). The resolving and stacking gels were effects involved in aggregation o f whey proteins to form
composed of 10% respo 4% acrylamide made up in TRIS/HC1 buffer, microparticles, more advanced methods are required, as
pH 8.45. The 1.5-mm-thick gels were run at a constant voltage of 200 V will be shown with chromatographic and electrophoretic
for 40 rain, stained by Coomassie Blue R 200 and destained for 12 h techniques. In Fig. 1 a typical elution profiles were depicted
and documented using the Gelimage Scanning System (Pharmacia).
comparing the patterns between Simplesse 100 and Lac-
prodan 80 under physiological conditions (0.2 mol/1 NaC1,
p H 6.9) on Sephacryl S-1000.
Results and discussion Both Simplesse 100 and Lacprodan 80 were resolved
with two peaks having m a x i m a close to 50 ml ( > 1 million
Solubility and structural features Da) and 125 ml ( < 100 kDa). Microparticles were eluted
within the first peak as a turbid fraction. The second peak
One o f the most intrinsic physical properties of proteins is was found to be [3-1actoglobulin and o~-lactalbumin in
their solubility. F o r the majority of proteins it is found best associated forms but also as monomers. Further extended
in the native status, whereas upon thermal processing most studies with different rates of protein denaturation revealed
proteins b e c o m e m o r e or less denatured. This is accom- a remarkable similarity between Simplesse 100 and heat-
panied by loss o f solubility. In Table 1 the N S I values were denatured whey protein (pH 6.5, 90~ for 10 rain) (not
c o m p a r e d for undenatured and denatured Lacprodan 80 as shown). F r o m these chromatographic findings it might be
well as for Simplesse 100 dry. concluded that both microparticulation and the generally
Results in Table 1 show that microparticulated protein is practiced whey protein denaturation would cause compa-
less soluble than Lacprodan 80 but more soluble than whey rable irreversible denaturations. However, it looked differ-
protein that has been thermally denatured at p H 6.5. ent in the presence of a disintegrating buffer like 3 mol/1
 291

A28o

1,ooo
=

0,500 i

go Igo ml 50 150 ml
Fig. 3. Elution profiles of supernatants (23,000 g130 min) (dotted line)
and sediments (solid line) of Lacprodan 80 (a) and Simplesse 100 (b)
on a Sephacryl S-1000 column (3 mol/1 urea, 20 mmol/1 imidazole,
50 mmol/1 NaC1, pH 6.9). The absorbances of all fractions were
measured at 280 nm (A2s0)

100 profiles, apparently most of the whey protein was

associated in microparticles, whereas the amount of solu-
ble protein remained low.
Fig. 2. SDS-PAGE of Simplesse 100 and Lacprodan 80 and their
chromatographicfractions obtainedby gel permeationchromatography
(data from Fig. l b). a, enriched [5-1actoglobulin; b, enriched a-
lactalbumin; c, Simplesse 100 1.5%; d, low molecular weight
(LMW) marker (Pharmacia); e, Lacprodan 80 fraction 2; f, Lacprodan The role of disaccharides
80 fraction 1; g, Simplesse 100 fraction 2; h, Simplesse 100 fraction 1;
i, Simplesse 100 1.5% The ability of lactose to protect milk protein against
thermocoagulation has been demonstrated by several work-
ers [7, 12, 13]. In practice it is comprehensible by the fact
urea, which was effective in dissolving Simplesse 100 that high-purity whey protein very often contains higher
(Fig. 1 b), but not heat-denatured Lacprodan 80. This may proportions of denatured protein compared with others
be causally related to non-covalent bonds in Simplesse 100 having higher lactose contents. Moreover, lactose may
favoured by the acidic denaturation step. Urea reduces the affect the mode and characteristics of whey protein aggre-
first peak in such a manner that the profile becomes more gates due to its ability to replace expelled water molecules
similar to that of Lacprodan 80 (Fig. 1 b). However, SDS in exposed hydrophobic pockets [13]. It is still an open
has been found to be effective also in reducing the first question as to whether and how lactose interacts with
peak, emphasizing that with microparticulation the disul- denatured protein under the conditions of microparticula-
phide mechanism does not play a role. tion. However, the good solubility of lactose proved to be a
Further characterization of the urea Sephacryl S-1000 hindrance in detecting or quantifying interactions between
fractions was made by SDS-PAGE (Fig. 2). In the first protein and lactose.
Simplesse 100 peak (50 ml) only ~-lactoglobulin was First attempts with Simplesse 100 using native PAGE
revealed, whereas the second peak (125 ml) contained combined with fuchsin staining failed to detect any protein-
both c~-lactalbumin and [3-1actoglobulin. The subtly differ- associated lactose with microparticles. So chromatographic
entiated presence of a-lactalbumin and [3-1actoglobulin in procedures were the methods of choice again with Sepha-
the Sephacryl S-1000 fractions might be of significance in dex G-100 or Sephacryl S-1000 followed by a colorimetric
the arrangement between non-covalent bonds. Assuming determination of lactose in the eluents. Results obtained
that the molecular contribution shown in Fig. 1 b concerns with Sephadex G-100 are shown in Fig. 4 a, b. These results
only the effects of split H bonds, then hydrophobic forces show that lactose remains associated with the protein to a
must be the other non-covalent forces responsible for the certain degree under physiological conditions. Both whey
molecular arrangement of ~3-1actoglobulin in the first peak. protein preparations contain lactose maxima within the first
The assumption is supported by the electrophoretic findings protein peak associated with aggregated whey protein.
obtained with SDS-PAGE (Fig. 2, lanes c, e, i), showing Lactose is also associated with free whey protein (165 ml
similar profiles for Lacprodan 80 and Simplesse 100. [3-1actoglobulin, 175 ml c~-lactalbumin). It seems that [3-
Extensive studies in solubility were made by separation lactoglobulin does have an increased affinity for lactose.
of diluted protein (1.5%) at 23.000 g for 30 rain. Both the In Fig. 5 the elution pattern of Simplesse 100 obtained
cleared supernatant as well as the sedimented protein were on Sephacryl S-1000 using 3 mol/1 urea buffer is shown.
investigated by gel permeation chromatography on Sepha- All the lactose is embedded in the last part of the second
cryl S-1000 in the presence of 3 mol/l urea. Results are peak, associated with o~-lactalbumin and [3-1actoglobulin as
shown in Fig. 3 a, b. Protein which remained soluble after shown above. It seemed that the embedded lactose might
the separation step is marked by dotted lines. Full lines signify a specific affinity with whey proteins eluting as the
indicate the sedimented protein. Considering the Simplesse second peak. Extended chromatographic studies with whey
292

A 280 A280 A 256

2,000.
O
1,000.
q

0,800.

60
1,000 0,600.
256 nm
............. 9 ....
40 e. . . . . . . . m . t ....
o 9

0,400
20 jr/
,:3 30 40 60 80 ~
. , , , ,

50 150 ml
A280
A 8o ,,.~ • n_.
1,000.
2,000
280 nm

"1 0,800'

................................
~ .e,,e
60 0,600'
1,000 256 nm

40

~
0,400.
-14t ,
20 b 30 40 60 80 ~C
Fig. 6. Effect of heat treatment on the absorbance at 280 n m (A280)or
256 nm (A2s6) of raw milk whey (a) and the same whey after a
5'0 150 rnl preliminary thermal modification (h) in the presence or absence of
Fig. 4. Elution profiles of Simplesse 100 (a) and Lacprodan 80 (b) on a 0.1% sucrose.
Sephadex G-100 column (0.2 tool/1 NaC1, pH 6.9) visualized by
absorbance at 280 nm (A280, dotted line) and lactose concentration
(gg lactose/ml, solid line) whey obtained by isoelectric precipitation of raw milk
and with the same whey after heating under acidic condi-
tions.
A 0,2
21~'lm
c:m

0,600 ~ :: 0.2
U V studies

oAoo

0,200

!
~i
"5o
ii
i
~

...................

lbo
!il
~,

/
!

l~o
0,1
Whey was heated to different temperatures ( 3 0 - 8 0 ~ C) for
1 rain, cooled with ice-water and centrifuged at 10,000 g
for 20 rain to remove any precipitate formed. The protein
concentration was estimated by the Bio-Rad procedure and
diluted to 0 . l % protein in 0.05% phosphate buffer. The
same experiment was repeated but in the presence of 0.1%
sucrose. The UV absorbance of the diluted whey samples
- ml
was read at 280 nm and 256 nm and plotted against
temperature. The results are shown in Fig. 6 a, b.
Fig. 5. Elution profile of Simplesse 100 on a Sephacryl S-1000 column
(3 tool/1 urea, 20 retool/1 imidazole, 50 retool/1 NaCI, pH 6.9) Heat-induced aggregations are mostly preceded by
visualized by absorbance at 280 nm (A280, dotted line) and lactose conformational changes in proteins and are detectable by
concentration (% lactose, solid line) increases in UV absorbance, like the exposure of tryptophan
residues visible at 280 nm, or the contribution of SH groups
at 256 nm [14, 15]. The results in Fig. 6 show that
proteins in the presence of increasing amounts of lactose carbohydrates promote conformational changes in whey
revealed that lactose eluted at the same position as shown protein. At 60~ the structural defolding reached a max-
with Simplesse 100 (Fig. 5), but describing a much wider imum with minimal denaturation (Fig. 6a). Increasing
amplitude. These results supported the assumption that temperature further to 70~ breaks the promotional effect
lactose stabilizes whey protein via non-specific preferen- of carbohydrates and whey protein becomes more and more
tial hydration. Related effects were observed with sour irreversibly denatured.
 293

These effects are not relevant to microparticulation References

because the process is preceded b y a thermal treatment
under acidic conditions. Related conformational changes 1. Singer SN, Yamamoto S, Latella J (1988) U.S. Patent 4.734.287
are best characterized b y UV readings at 30~ (Fig. 6b). 2. Kammerlehner J (1993) dmz-Lebensmittelind Milchwirtsch 114:
848
Heating under acidic conditions caused a high degree of
3. Lieske B, Konrad G (1993) Dtsch Milchwirtsch 44:1252
molecular defolding which is stabilized by the hydroxyl 4. Harwalkar VR (1979) Milchwissenschaft 34:419
groups o f lactose as reported with ovalbumin [16]. At 5. Modler HW, Harwalkar VR (1981) Milchwissenschaft 36:537
temperatures higher than 65~ a surplus of carbohydrate 6. Modler HW, Jones JD (1987) Food Technol 41:114
affects the thermocoagulation b y smoothing out the sharp 7. Garrett JM, Stairs RA, Annett RE (1988) J Dairy Sci 71:10
transition between 65~ and 70 ~ C. It is conceivable that an 8. Morr CV, German B, Kinsella JE, Regenstein JM, Van Buren JR
Kilara A, Lewis BA, Mangino ME (1985) J Food Sci 50:1715
increased thermoflexibility might be advantageous to a
9. Jelen R Buchheim W (1984) Milchwissenschaft 39:215
more controlled aggregation o f whey protein. 10. Lieske B (1972) Milchforsch Milchprax 14:21
11. Sch~iggerH, von Jagow G (1987) Anal Biochem 166:368
Acknowledgements. We are grateful to the German Federal Govern- 12. Plock J, Kessler HG (1992) dmz-Lebensmittelind Milchwirtsch
ment for supporting us within the Scientists Integration Programme for 113:895
East-German scientists. 13. Jost R (1993) Trends Food Sci Technol 4:283
14. Anfinsen CB, Scheraga (1975) Adv Protein Chem 29:205
15. Ewbank JJ, Creighton TE (1993) Biochemistry 32:3694
16, Simpson RB, Kautzmann W (1953) J Am Chem Soc 75:5139

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