Towards recombinantly produced milk proteins: Physicochemical and emulsifying properties of engineered whey protein beta-lactoglobulin variants [original]

Food Hydrocolloids 110 (2021) 106132

Available online 7 July 2020

Towards recombinantly produced milk proteins: Physicochemical and

emulsifying properties of engineered whey protein

beta-lactoglobulin variants

Julia K. Keppler

, Anja Heyse

, Eva Scheidler

, Maximilian J. Uttinger

, Laura Fitzner

Uwe Jandt

, Timon R. Heyn

, Vanessa Lautenbach

, Joanna I. Loch

, Jonas Lohr

Helena Kieserling

, Gabriele Günther

, Elena Kempf

, Jan-Hendrik Grosch

Krzysztof Lewi�

nski

, Dieter Jahn

, Christian Lübbert

, Wolfgang Peukert

, Ulrich Kulozik

Stephan Drusch

, Rainer Krull

, Karin Schwarz

, Rebekka Biedendieck

Wageningen University, Laboratory of Food Process Engineering, P.O. Box 17, 6700 AA, Wageningen, the Netherlands

Kiel University, Division of Food Technology, 24118, Kiel, Germany

Technische Universit€

at Berlin, Chair of Food Technology and Food Material Science, Straße des 17. Juni 135, 10623 Berlin, Germany

TU Munich, Chair of Food and Bioprocess Engineering, Weihenstephaner Berg 1, 85354, Freising, Germany

Friedrich-Alexander-Universit€

at Erlangen-Nürnberg, Institute of Particle Technology, Interdisciplinary Center for Functional Particle Systems, Haberstraße 9a, 91058

Erlangen, Germany

Hamburg University of Technology, Bioprocess and Biosystems Engineering, Hamburg, Germany

Deutsches Elektronen-Synchrotron DESY, Information Technology, Notkestrasse 85, 22607, Hamburg, Germany

Jagiellonian University, Faculty of Chemistry, Gronostajowa 2, 30-387 Krak�

ow, Poland

Technische Universit€

at Braunschweig, Institute of Biochemical Engineering, Rebenring 56, 38106, Braunschweig, Germany

Technische Universit€

at Braunschweig, Center of Pharmaceutical Engineering (PVZ), Franz-Liszt-Straße 35a, 38106, Braunschweig, Germany

Technische Universit€

at Braunschweig, Braunschweig Integrated Centre of Systems Biology (BRICS), Rebenring 56, 38106, Braunschweig, Germany

Technische Universit€

at Berlin, Chair of Food Colloids, Straße des 17. Juni 135, 10623 Berlin, Germany

Technische Universit€

at Braunschweig, Institute of Microbiology, Rebenring 56, 38106, Braunschweig, Germany

1. Introduction

The growing world population and its increasing demand for high-

quality protein will have a strong impact on future research topics.

The production of animal proteins as sole protein source is not sus-

tainable because it takes up enormous environmental impact with low

production efficiency. Various approaches are already aimed at

addressing these challenges: Alternative and sustainable sources such as

plants, insects, fungi and bacteria are being tested for their natural

amino acid value and protein yield (Dekkers, Boom, & van der Goot,

2018; Mishyna, Chen, & Benjamin, 2020; van der Weele, Feindt, van der

Goot, van Mierlo, & van Boekel, 2019). In order to increase consumer

Abbreviations: BLG AB, bovine beta-lactoglobulin isoform AB mixture; BLG B, bovine beta-lactoglobulin isoform B; rBLG B, recombinant bovine beta-lactoglobulin

isoform B with additional N-terminal M; sBLG B, recombinant bovine beta-lactoglobulin isoform B with N-terminal L1A/I2S substitutions; AUC, analytical ultra-

centrifugation; DLS, dynamic light scattering; DNPH, 2,4-dinitrophenylhydrazine; DiTyr, dityrosine; DTNB, 5,5-’Dithio-bis-(2-nitrobenzoic acid); DTT, Dithiothreitol;

Gua-HCl, guanidine hydrochloride; OPA, ο-phtalaldehyde; PMSF, phenylmethylsulfonylfluoride; PTM, post-translational modifications; NAC, N-acetyl-

L-cysteine;

NFK, N-formylkynurenine.

* Corresponding author. Wageningen University & Research, AFSG: Laboratory of Food Process Engineering, P.O. Box 17, 6700 AA Wageningen, Wageningen

Campus, Building 118 (Axis), Bornse Weilanden 9, 6708 WG, Wageningen, the Netherlands.

E-mail addresses: [email protected] (J.K. Keppler), [email protected] (A. Heyse), [email protected] (E. Scheidler), [email protected]

(M.J. Uttinger), [email protected] (L. Fitzner), [email protected] (U. Jandt), [email protected] (T.R. Heyn), [email protected]

(V. Lautenbach), [email protected] (J.I. Loch), [email protected] (J. Lohr), [email protected] (H. Kieserling), g.guenther@tu-

braunschweig.de (G. Günther), [email protected] (E. Kempf), [email protected] (J.-H. Grosch), [email protected]

(K. Lewi�

nski), [email protected] (D. Jahn), [email protected] (C. Lübbert), [email protected] (W. Peukert), [email protected]

(U. Kulozik), [email protected] (S. Drusch), [email protected] (R. Krull), [email protected] (K. Schwarz), r.biedendieck@tu-

braunschweig.de (R. Biedendieck).

Contents lists available at ScienceDirect

Food Hydrocolloids

journal homepage: http://www.elsevier.com/locate/foodhyd

https://doi.org/10.1016/j.foodhyd.2020.106132

Received 22 March 2020; Received in revised form 8 June 2020; Accepted 24 June 2020

Food Hydrocolloids 110 (2021) 106132

acceptance, these proteins are also selected for their ability to mimic the

texture and taste of meat or dairy products (Dekkers et al., 2018; Paul,

Kumar, Kumar, & Sharma, 2019; Sethi, Tyagi, & Anurag, 2016).

Another approach is the possibility of in vitro farming (cellular

agriculture). Recombinant production of chymosin for cheese making

has been done for years (Tagliavia & Nicosia, 2019). Novel approaches

are the in vitro production of meat via tissue engineering (Bhat, Kumar,

& Fayaz, 2015). Further, there is potential interest in recombinantly

produced dairy proteins (Vestergaard, Chan, & Jensen, 2016). The

commercial and environmental sustainability of these recombinantly

produced proteins/products at a scale relevant for feeding the world

population has still to be proven. In order to clarify the challenges and

opportunities of this emerging technology, it is of great importance to

investigate the production processes and functional equivalence of re-

combinant proteins.

The production of recombinantly produced whey protein beta-

lactoglobulin (BLG) is particularly interesting in this respect, as it is

the main protein in bovine whey and as such an important component in

many foods. It also has versatile functional properties, e.g. as gelling,

foaming and emulsifying agent (Dombrowski, Gschwendtner, Saalfeld,

& Kulozik, 2018; Keppler, Steffen-Heins, Berton-Carabin, Ropers, &

Schwarz, 2018; Lam & Nickerson, 2014). Since BLG is also a central

model protein in structural biology, several efforts for its recombinant

production have been published. It exists predominantly in two isoforms

(BLG A and BLG B) in bovine milk, which differ by two amino acids.

Many studies focused on BLG A or B production in yeasts (Denton et al.,

1998; Kim et al., 1997; Yagi, Sakurai, Kalidas, Batt, & Goto, 2003),

which resulted in a high yield but either contained several changes in the

N-terminal region of BLG and/or led to co-produced (poly)saccharides.

The latter can interact with BLG and lead to structural changes as well as

protein-protein interactions (Hundschell, B€

ather, Drusch, & Wagemans,

2020). Yeast-based production and commercial application of recom-

binant BLG for food products are described in patent US9924728B2 et al.

(2018) and Perfect Day Inc. started to commercialize. The company

admits that their product may exhibit post-translational modification

(PTM) in the form of glycozylation, which is typical for proteins

recombinantly produced in eukaryotes.

However, a correctly folded protein with high amino acid avail-

ability, preserved functionality, and solubility is essential to replace the

natural BLG in a broad range of food applications. Batt, Rabson, Wong,

and Kinsella (1990) proposed the intracellular production of BLG in

Escherichia coli (E. coli) as a prelude for potential structure/function

related research through site-directed mutagenesis, which initially led to

insoluble protein. Ponniah and colleagues finally succeeded in produc-

ing correctly folded soluble BLG B in E. coli (Ponniah et al., 2010) still

carrying the N-terminal start methionine (rBLG B) which is missing in

the native bovine BLG B. This variant has recently been further

improved by Loch et al. by introducing a L1A/I2S mutation (sBLG B) to

ensure correct cleavage of the N-terminal methionine (Loch et al., 2016),

which was suitable for ligand binding studies. In addition, protein

isolation from the microorganism has been optimized to remove the

endogenous fatty acids that were bound in the hydrophobic protein

pocket after protein production as well as to ensure high purity and

nativity. The crystal structure and conformation of the recombinant

sBLG B (PDB ID: 6QI6) produced in this way is very similar to that of the

natural bovine BLG B (PDB ID: 3NPO), isolated from bovine milk (Loch

et al., 2016) and the recombinant protein was considered suitable for

biochemical studies.

Now it is also of interest to study the possibility to use these proposed

genetically engineered BLGs from E. coli with respect to possible food

applications. Here, a recombinant wild type BLG B variant still carrying

a methionine at its N-terminus (rBLG B) but without any further amino

acid mutations as well as a recombinant BLG B variant with L1A/I2S

modification (sBLG B) for accurate N-terminal cleavage of the methio-

nine proposed by Loch et al. (2016) were produced in E. coli and purified

with an adapted protocol. The physicochemical and emulsifying

properties of the recombinant variants were compared with those of

commercial BLG B isolated in high purity from bovine milk. In addition,

isolated BLG AB was chosen as reference, because this mixed variant is

present in all food-related BLG formulations. Similar emulsifying prop-

erties (in the range of commercial BLG B and isolated BLG AB) are an

important prerequisite for the substitution of bovine BLG with recom-

binant BLG in many food products.

Our approach is new and unique in that it investigates how the

necessary/unavoidable N-terminal modifications of the recombinant

BLG variants affect their physicochemical and functional properties in

the context of potential food applications, in particular, their emulsion-

forming capacity. This work differs from previous work in the field as it

is intended to investigate whether the emulsion forming ability of the

recombinant variants is equivalent to that of BLG isolated from bovine

milk while many previous studies with recombinant BLG rather focused

on structure/functionality effects of cysteine 121 mutagenesis or addi-

tion of a third disulphide bond (Jayat et al., 2004; Cho et al., 1994). The

working hypothesis of the present investigations is that the small

structural changes that were necessary/unavoidable for the expression

of a correctly folded recombinant BLG B in E. coli might modify some

physicochemical properties but not affect its functionality (i.e., emulsi-

fying properties) in the final product.

The results presented here provide the possibility of a better under-

standing of the behavior of the genetically modified BLG variants under

processing induced stress and their functionality compared to those of

native bovine BLG. This manuscript also gives a comprehensive over-

view of the physicochemical properties of different BLG variants,

modeled or measured with a large variety of current methods, as well as

insights into the functionality of two different recombinant variants with

specific single amino acid changes. This provides the basis for evaluating

the substantial equivalence of the recombinant variants as a prerequisite

for approval in the food industry. It also allows the selection of the most

suitable recombinant variant for potentially high-yield production also

in other bacteria than E. coli, to eventually be used in food products. In

the future, such results may contribute to the assessment of economic

and ecological sustainability of this concept.

2. Materials & methods

2.1. Materials

The recombinant BLG B variants were produced and isolated as

described further below. Commercial protein BLG B (L8005, isolated

from bovine milk) was purchased from Sigma Aldrich (Steinheim, Ger-

many). Bovine BLG AB was isolated from bovine milk according to

Toro-Sierra, Tolkach, and Kulozik (2013). 5,5-’Dithio-bis-(2-ni-

trobenzoic acid) (DTNB), ο-phtalaldehyde (OPA), 2,2-Bis(hydrox-

ymethyl)-2,20,200-nitrilo-triethanol, L-leucine, Dithiothreitol (DTT), 2,

4-dinitrophenylhydrazine (DNPH) and phenylmethylsulfonylfluoride

(PMSF) were purchased from Sigma Aldrich (Steinheim, Germany).

Boric acid, glycine, N-acetyl-L-cysteine (NAC), Tris, sodium hydroxide

(NaOH), o-phosphoric acid (85%), acetonitrile (ACN), guanidine hy-

drochloride (Gua-HCl), trifluoroacetic acid (TFA), trisodium citrate,

hydrochloric acid (HCl, 37%) and Florisil (activated magnesium silicate;

MgO ⋅ 3.6 SiO

⋅ 1.53 OH, 100%) was from Carl Roth (Karlsruhe, Ger-

many), Merck (Darmstadt, Germany), and/or Sigma Aldrich (Steinheim,

Germany). Benzonase (250 U/

L) was purchased from Merck (Darm-

stadt, Germany). Ethanol and ethyl acetate were from VWR (Radnor,

USA). Middle-chain triacylglyceride oil (Witarix MCT 60/40, >99.9%)

was purchased from IOI Oleochemical (Hamburg, Germany). All

chemicals were of analytical grade. For all experiments, ultrapure water

(�18.2 MΩ cm

1

) was used. Borosilicate membranes (type P4) were

purchased from ROBU Glasfilter-Ger€

ate (Hattert, Germany).

J.K. Keppler et al.

Food Hydrocolloids 110 (2021) 106132

2.2. Sample preparation

Unless otherwise stated, protein solutions were prepared by dis-

solving 10 mg/mL commercial BLG B, isolated bovine BLG AB, or one of

the two recombinant variants rBLG B and sBLG B in ultra-pure water.

After stirring the protein solutions for at least 1 h, the pH value was set to

pH 7 using NaOH.

2.3. Production and purification of recombinant BLG

2.3.1. Recombinant production

For the production of recombinant bovine BLG B, E. coli Origami B

(DE3) cells (Novagen, Darmstadt Germany) were transformed with the

plasmids pETDuet-1/DsbC/BLGB (encoding recombinant beta-

lactoglobulin isoform B, denoted as “rBLG B00) and pETDuet-1/DsbC/

L1A/I2S-BLGB (encoding recombinant beta-lactoglobulin isoform B

with modified N-terminus L1A/I2S, denoted as “sBLG B00), respectively,

as described previously (Loch et al., 2016). Recombinant cells were

grown in 50 mL LB-medium supplemented with 100

g/mL carbenicillin

and 2 g/L glucose at 37 �C and 150 min

1

overnight. 44 mL of this

culture were added to 1 L LB-medium supplemented with 100

g/mL

carbenicillin and 2 g/L glucose in a 2 L baffled shaking flask and incu-

bated until reaching an OD

600

of 0.5–0.7 (pETDuet-1/DsbC/BLGB) and

1.0–1.2 (pETDuet-1/DsbC/L1A/I2S-BLGB), respectively. Recombinant

protein production was induced by adding 500

M iso-

propyl-β-D-thiogalactopyranosid (IPTG, Sigma-Aldrich). After incuba-

tion for 4 h (pETDuet-1/DsbC/BLGB) and overnight

(pETDuet-1/DsbC/L1A/I2S-BLGB), respectively, at 25 �C and 140

min

1

, cells were harvested at 4 �C (20 min, 3000 g), resuspended in 20

mL sodium phosphate buffer (20 mM, pH 6), harvested again at 4 �C (40

min, 3000 g) and stored at 80 �C. 1 L cell culture resulted in approx-

imately 4.5–8 g of wet cells.

2.3.2. Purification

For the purification of recombinant BLG B variants, 30 mL sodium

phosphate buffer (20 mM, pH 6) was added and cells were thawed on

ice, resuspended, and supplemented with 15

L PMSF (200 mM) and

0.15

L benzonase (250 U/

L) per 30 mL. Cells were disrupted by son-

ication for 4 ⋅ 5 min, with 5 min gaps between (settings: 97% power, 5 ⋅

10% cycles). Disrupted cells were transferred into 2 mL reaction vials

and centrifuged for 90 min at 18,000 g and 8 �C. The supernatant was

filtrated (pore size of 0.45

m). Before loading the filtrated cell lysate

(around 3000 mg total protein) on a HiTrap Capto Q ImpRes chroma-

tography column (10 mL column volume; GE Healthcare Life Sciences,

Chicago, USA), the anion exchange column was equilibrated with so-

dium phosphate buffer (20 mM, pH 6). Proteins were eluted using a

linear gradient from zero to 500 mM NaCl (20 column volumes). Frac-

tions containing the highest amount of recombinant BLG B were com-

bined, adjusted to 1.5 M of ammonium sulfate, incubated overnight at 4

�C and centrifuged for 20 min at 2900 g and 4 �C. 7.5 mL of the su-

pernatant containing BLG B was loaded on a HiPrep desalting 26/10

column (53 mL column volume; GE Healthcare Life Sciences, Chicago,

USA) and eluted using ultrapure water. Finally, proteins were lyophi-

lized for 62 h using a temperature gradient from 35 �C to 25 �C (Beta

2–8 LSCplus, Martin Christ, Osterode am Harz, Germany). Vials con-

taining lyophilized BLG B were closed under dry nitrogen atmosphere

and stored at 4 �C. All chromatographic purification steps were carried

out using the €

Akta Pure 150 (GE Healthcare Life Sciences, Chicago, USA)

fast protein liquid chromatography system. Throughout the whole pu-

rification process, protein fractions were analyzed by SDS-PAGE (12%

(w/v) of acrylamide) as described before (Righetti, 1990).

2.4. Sequence and structure analyses

BLG concentrations were measured by NanoDrop microvolume

spectrophotometer (Peqlab Biotechnology GmbH, Erlangen, Germany).

In addition, the percentage of the BLG protein band in comparison to all

bands was calculated from the SDS-Page images using GelAnalyzer

software (version 19.1, free software, GelAnalyzer.com). The N-terminal

amino acid sequence of the recombinant variants was analyzed by

automatic Edman degradation (Edman & Begg, 1967).

2.4.1. Analysis of covalent protein modifications

Mass spectrometry was conducted to screen for acetylation, glyco-

zylation, or other covalent protein modifications occurring in bovine

milk or during recombinant production. In addition, mass shifts reveal

changes in the amino acid sequence. For the analysis of the protein

samples a Qq-TOF (Bruker, Impact II, Resolution 40.000) with ion-

mobility analysis (Seadm, differential mobility analysis, DMA, Resolu-

tion 60) was used.

2.4.2. Molecular dynamic analysis

The BLG variants were analyzed using molecular dynamics (MD),

which gives information on the dynamic properties of the protein vari-

ants. All MD simulations and analyses were performed using the GRO-

MACS software package version 5.1.1 (Van Der Spoel et al., 2005) and

the OPLS-AA (all-atom) force field (Kaminski, Friesner, Tirado-Rives, &

Jorgensen, 2001). The structure of native dimeric BLG has been ob-

tained from PDB 3PH5. The original structure misses a few N-terminal

amino acids, i.e., it starts at a position denoted there as number 20

(TQTMK–; c.f. Table 1). The remaining N-terminal amino acids for all

variants (BLG A or B: LIV–; rBLG B: MLIV–; sBLG B: ASV–) were added

Table 1

Sequence and structure properties of BLG variants used within this study.

Name BLG AB BLG B rBLG B sBLG B

Purity (Nanodrop) [%] >90 >90 >90 >90

Sequence of N-Terminus LIVTQ LIVTQ MLIVTQ ASVTQ

Length [amino acids] 162 162 163 162

Calculated molecular

weight [Da]

18,366.27 18,280.17 18,411.36 18,211.99

MS measured molecular

weight [Da]

18,374.9 18,288.9 18,419.9 18,220.9

MS measured mass

difference to BLG B

[Da]

þ86 0 þ131 68

Measured SH groups

[mol/mol protein]

0.43 �

0.0

b,c

0.35 �0.0

0.45 �0.0

0.38 �

0.0

a,b

Measured NH groups

[mol/mol protein]

19.6 �

0.5

17.6 �

1.0

a,b

17.0 �0.1

16.7 �0.6

Measured isoelectric

point (IEP)

4.9 �0.12 4.7 �0.20 5.4 �0.05 5.4 �0.08

Theoretical IEP

calculated for

unfolded BLG

4.76 4.83 4.83 4.83

Acid solubility [%] 98 �0.6

88 �0.4

96 �0.3

97 �0.5

a,b

MD simulations

MD root mean squared

deviation, all atoms

[nm]

n. d. 0.29 �

0.022

0.36 �

0.068

0.29 �

0.022

MD radius of gyration of

monomer [nm]

n. d. 1.53 �

0.013

1.54 �

0.016

1.52 �

0.013

MD SASA of single

monomers [nm

]

n. d. 178.9 �

1.4

183.0 �

1.75

178.5 �

1.53

MD SASA of dimer

[nm

]

n. d. 166.2 �

1.0

168.9 �

1.10

165.6 �

1.01

MD intramolecular H-

bonds of monomer

n. d. 224 �3.1

225 �3.7

223 �3.0

MD intramolecular H-

bonds of dimer

n. d. 231 �4.8

232 �4.3

234 �3.4

Properties of BLG A are listed instead of BLG AB for some instances where only

monomer structure was assessed. Significant differences in a row (p <0.05) are

indicated by small letters (a-c). MD simulations of BLG AB were not determined

(n. d.) because of its presence as a homodimer (AA or BB) or heterodimer (AB) in

a mixture at pH 7. H-bonds, hydrogen bonds; MD, molecular dynamics; MS, mass

spectrometry; SASA, solvent accessible surface area.

J.K. Keppler et al.

Food Hydrocolloids 110 (2021) 106132

using Modeller V9.15 (Marti-Renom et al., 2000). The resulting models

were equilibrated and simulated as described in Uttinger et al. (2020).

The temperature was maintained at T ¼293 K, pH was set neutral and

salt concentration was set to 10 mM. Two types of simulations were

performed subsequently: First, 5 ns of free (unforced) simulation was

conducted (MD-free), followed by 0.7 ns of forced simulation, separating

the two monomers along the dimer’s principal axis by pulling with

constant velocity (v ¼5 ms

1

) and adapted force (MD-pull). Root mean

squared deviations of respective structures (RMSD), radius of gyration

), surface accessible area (SASA), hydrogen bonds binding energies

were extracted for all simulations and pulling forces from the MD-pull

simulations using the respective GROMACS (GROningen MAchine for

Chemical Simulations) tools. Each simulation was repeated indepen-

dently, yielding n ¼20 replicates for each variant, thus total 120 sim-

ulations. Comparison of extracted data between variants allowed for

statistical significance testing (Student’s two-sided t-test) and calcula-

tion of confidence intervals (p ¼95%, assuming normal distribution).

2.4.3. Analysis of free thiol group reactivity (RSH test)

The number of accessible thiol groups (SH groups) per protein was

determined according to a variation of the Ellman’s assay (Ellman,

1959) without denaturing conditions as reported previously (Keppler

et al., 2014). 1.6 mL tris-glycine buffer (50 mM, pH 8.5), 400

L BLG

solution, and 40

L DTNB (10 mM in 50 mM tris-glycine buffer, pH 8.5)

were mixed and incubated for 10 min at room temperature. The

absorbance of the mixture was measured at 412 nm using a spectro-

photometer (Helios Gamma, UV–Vis, Thermo Spectronic, Cambridge,

UK). The concentration of free thiol groups, expressed as free thiol

groups per protein molecule, was determined using the molar attenua-

tion coefficient of 13,600 M

1

. All results were corrected for the

protein concentration which was determined by measuring the absor-

bance of the supernatant at 278 nm after centrifugation using a molar

extinction coefficient of 17,600 M

1

2.4.4. Analysis of free amino groups (NH groups)

The amount of accessible amino groups on the different BLG variants

was measured with the ο-phtalaldehyde (OPA) method. BLG concen-

trations were set to 3.33 mg/mL (~180

M). Due to poor solubility,

rBLG B was centrifuged for 5 min at 4000 g and 20 �C. All results were

corrected for the protein concentration which was determined by

measuring the absorbance of the supernatant at 278 nm using a molar

extinction coefficient of 17,600 M

1

The determination of free amino groups was performed according to

Roth (Roth, 1971) with slight modifications. In the presence of reduced

thiol groups, OPA reacts with free amino groups by forming a fluo-

rophore. 120

L protein solution was mixed with 2.64 mL NAC (18.4

mM) in borate buffer (100 mM, pH 9.3) and 150

L ultra-pure water

instead of a denaturing agent. The mixture was incubated at 50 �C for 10

min. After the addition of 90

L OPA (250 mM) the mixture was further

incubated at 50 �C for 30 min. The final protein concentration was

approximately 5

M. The mixture was cooled down to room temperature

for 30 min and the absorbance was measured at 340 nm using a spec-

trophotometer (Helios Gamma, UV–Vis, Thermo Spectronic, Cambridge,

UK). For the calculation of the amount of free amino groups, a standard

curve of L-leucine in a concentration range of 1.2–120

M was used. The

amount was expressed as the number of free amino groups per protein

molecule.

2.4.5. Zeta potential and isoelectric point

The zeta potential is related to the net surface charge of the proteins

and was measured using the Zetasizer instrument Nano ZS (Malvern

Instruments, Herrenberg, Germany). The solutions were dialyzed

against 10 mM NaCl solution for at least 4 h, and the dialysate was

changed at least once to remove minor contamination. The pH of the

protein solutions was adjusted with HCl and NaOH. BLG concentrations

were set to 1.0 mg/ mL (~60

M). The samples were prepared using a

similar 10 mM NaCl solution as used in dialysis in order to ensure

constant ionic strength after dilution, which is important for the sub-

sequent analytical ultracentrifugation (AUC) analysis for a determina-

tion of the dimer dissociation constants K

. Finally, the protein

concentrations were controlled again via UV/VIS spectroscopy (Analytic

Jena, Specord 210, Jena, Germany). The pH was controlled directly

before the measurements and adjusted accordingly using HCl and

NaOH.

For the determination of the isoelectric point (IEP), different pH

values were set between 2 and 11 with 100 mM NaOH and 100 mM HCl

to determine the point where the zeta potential reaches 0. For all mea-

surements, a folded-capillary cuvette (Malvern Instruments, Malvern,

Grovewood, UK) was used. The temperature was controlled at 25 �C

with an initial equilibration time of 120 s.

2.4.6. Acid solubility by HPLC

To determine the relative amount of correctly folded BLG of each

variant, the reversed phase (RP)-HPLC method to determine whey

proteins in cow’s milk was applied, modified according to Dumpler,

Wohlschl€

ager, and Kulozik (2017). In brief, BLG concentrations were set

to 1 mg/mL and stirred at room temperature for 4 h. To determine the

native BLG content, the pH of the protein solutions was adjusted to 4.6

reaching the isoelectric point of denatured BLG, which facilitates their

precipitation, while intact BLG remains soluble in the supernatant. The

concentration of denatured BLG was calculated from the content in the

supernatant relative to the amount in the original sample.

For analysis, 200

L of each sample was mixed with 800

L of buffer

and incubated for 30 min at room temperature. The mixture was

manually shaken to ensure the complete solubilization of proteins.

Analysis was carried out using an Agilent 1100 series chromatograph

(Agilent, Waldbronn, Germany) equipped with a Latek 300 Å PLRP-S-

C18 (Agilent, Waldbronn, Germany) column and eluted with a

gradient of the two eluents A and B. Eluent A contained 90% ultrapure

water, 10% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA),

eluent B contained 10% ultrapure water, 90% ACN and 0.07% TFA. The

RP-HPLC was performed with a flow rate of 1 mL/min with the

following gradient of water and ACN: 0–2 min (43–47% B), 6–9 min

(49–52% B), 11–13 min (54–55%), 13.5–13.8 min (100%) and 14 min

(100–14% B). The injection volume was 20

L. The temperature of the

column was 40 �C. The proteins were detected by UV/Vis at a wave-

length of 226 nm. The evaluation of chromatograms was performed

using the Agilent ChemStation Software (Rev.B.04.03.). Experiments

were performed in duplicate.

2.4.7. Conformation analysis by FTIR

Protein secondary structure was analyzed using ATR-Fourier trans-

form infrared spectroscopy (FTIR) on a Confocheck™ Tensor II system

(Bruker Optics, Ettlingen, Germany) optimized for protein analytics in

solutions and a thermally controlled BioATR2 unit as previously

described by Kayser, Arnold, Steffen-Heins, Schwarz, and Keppler

(2020).

Measurements were conducted at a temperature of 25 �C against the

respective solvent mixtures without protein as background and averaged

over 120 scans at a resolution of 0.7 cm

1

. For evaluation, the measured

spectra underwent atmospheric correction and were then vector-

normalized. The second derivative of the amide band І (1590 - 1700

1

) was calculated using nine smoothing points.

2.4.8. Conformation analysis by intrinsic tryptophan fluorescence

Conformational differences between the BLG variants were investi-

gated using intrinsic tryptophan fluorescence. The analysis was con-

ducted as previously described (Heyn et al., 2019): The intrinsic

tryptophan fluorescence of centrifuged samples was carried out in

quartz cuvettes using a Varian Cary Eclipse fluorescence spectropho-

tometer (Varian Darmstadt, Germany) with protein concentrations of

250 mg/mL. All measurements were performed at pH 7 with an

J.K. Keppler et al.

Food Hydrocolloids 110 (2021) 106132

excitation of 295 nm. Emission spectra were collected between 310 and

404 nm. The results were corrected by the actual protein concentration,

which was measured by the absorbance at 278 nm as described above.

2.5. Quaternary structure analysis

2.5.1. Molecular weight distribution in solution by analytical

ultracentrifugation (AUC)

AUC follows the sedimentation velocity of proteins, which differ for

different quaternary structures. BLG concentration series (0.25mg/mL

to 1.5 mg/mL) were produced from a stock solution of 1.5 mg/mL .

Therefore, BLG variants were dialyzed as described for zeta potential

and IEP measurements.

A modified preparative centrifuge (Type Optima L-90 K, Beckman

Coulter, Krefeld, Germany) was used for the sedimentation velocity (SV)

AUC experiments. The experiments were performed at a fixed rotor

speed of 50,000 min

1

. The temperature was set to 20 �C throughout the

measurements. Titanium centerpieces with a path length of 12 mm were

used for all experiments. The individual SV data, which are collected for

each protein concentration and solution pH were analyzed with the

continuous c(s)-model of the SEDFIT program to determine sedimenta-

tion coefficient distributions and the molar masses of the individual

species (Brown & Schuck, 2006). Determination of the dimer dissocia-

tion constant K

was conducted from a protein concentration series at

preset pH of the solution and analysis of the respective sedimentation

data was carried out via SEDANAL program (version 6.93) (Stafford &

Sherwood, 2004). The equilibrium constant K

is derived from the

protein monomer-dimer equilibrium reaction and describes the ratio of

association to dimers and dissociation to monomers. Further details are

included in the supplementary. The partial specific volume for all BLG

samples was set to 751

L/g. The solvent properties were equivalent to

the properties of water for all analysis steps. Concentration non-ideality

effects are in these concentration ranges not interfering with these

measurements (Uttinger et al., 2019).

2.5.2. Hydrodynamic diameter by dynamic light scattering (DLS)

In order to assess the hydrodynamic diameter of the different BLG

variants, protein size was measured with a Zetasizer instrument (Nano

ZS, Malvern Instruments, Herrenberg, Germany). Protein powders were

solubilized in PBS (137 mM NaCl, 2.7 mM KCL, 10 mM Na

HPO

, 1.8

mM KH

) at a concentration of 1 mg/mL in order to assure correct

DLS analysis and the pH was adjusted to 7.0. The measurement was

carried out at 20 �C. If necessary, protein solutions were filtered through

a 0.45

m cellulose filter. Then to track the particle size, 10 measure-

ments were conducted. Each measurement was performed over a time

span of 60 s. Measurements were carried out in backscattering mode at

173�to achieve the highest resolution for smaller particles. A refractive

index of 1.45 was chosen and the solvent viscosity was assumed to be

similar to water. The experiments were conducted in triplicate.

2.6. Physicochemical properties

2.6.1. Denaturation temperature by differential scanning calorimetry

(DSC)

In order to determine the denaturation temperature of the proteins,

differential scanning calorimetry was performed using a TA instruments

Q 1000 series (TA instruments, Eschborn, Germany). BLG concentra-

tions were set to 100 mg/mL (w/w) at pH 7 and incubated at room

temperature for 4 h. Prior to the analysis, the instrument was equili-

brated at 25 �C for 2 min. Then, the modulated (0.5 �C/min) ramp from

25 to 95 �C was started with a heating rate of 2 �C/min. After completing

the ramp, the temperature was kept at 95 �C for 1 min. Experiments

were performed in duplicate.

2.6.2. Protein hydrophobicity by HPLC

RP-HPLC was used as a measure of the protein hydrophobicity by

applying a similar HPLC method as the one used for the acid solubility

with minor variations: BLG concentrations were set to 1 mg/mL with

ultra pure water and analyzed by RP-HPLC as previously described

(Keppler et al., 2017). Shortly, RP-HPLC analysis was carried out on a

Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific, Waltham,

USA) equipped with a polymeric reversed phase column (PLRP-S, 300 Å,

m, 150 ⋅ 4.6 mm, Agilent Technologies, Santa Clara, USA). The

separation was performed using ultra pure water with 0.1% TFA as

eluent A and ACN with 0.1% TFA as eluent B. The applied gradient was

0–1 min (35% B), 8 min (38% B), 16 min (42% B), 22 min (46% B),

22.5–23 min (100% B) and 23.5 min (35% B). The injection volume was

L at a flow rate of 1 mL/min. The temperature of the column was 40

�C. The detection wavelength was 205 nm.

In addition, the samples were also analyzed in a reducing and

denaturating buffer system (0.02 M DTT and 6 M Gua-HCl) using the

same column, flow rate, and column temperature, but detection wave-

length of 226 nm and slightly different eluents mixtures: Eluent A: 100%

ultrapure water, 0.1% TFA and Eluent B: 20% ultrapure water, 80%

ACN, 0.0555% TFA. The applied gradient was 0 min (43% B), 2 min

(47% B), 6 min (49% B), 9 min (52% B), 11 min (54% B), 13 min, (55%

B), 13.5–13.8 min (100% B) and 14 min, (43% B).

2.7. Protein oxidation (carbonyl content, NFK, dityrosine)

Protein oxidation can occur during protein purification from milk or

bacterial suspension. Protein carbonyl content was analyzed as

described by Levine et al. (1990) and Scheidegger, Pecora, Radici, and

Kivatinitz (2010). BLG concentrations were set to 3.33 mg/mL (~180

M). Due to poor solubility, the recombinant wild type (rBLG B) was

centrifuged for 5 min at 4000 g and 20 �C. For each sample, two aliquots

were taken and analyzed as pairs (A and B): The protein (2 mg) was

precipitated in each sample using 1 mL TCA (20%) and harvested by

centrifugation at 10,000 g and room temperature for 5 min. The su-

pernatant was discarded. One mL DNPH (100 mM in 2 M HCl) was

added to one precipitate (A) and 1 mL HCl (2 M) the other (B). After

incubation in the dark for 1 h at room temperature, 800

L TCA (20%)

were added, the samples were vortexed and centrifuged at 10,000 g for

5 min. The precipitated proteins were washed 3 times with 1 mL ethyl

acetate/ethanol (1:1, v/v) to remove unbound DNPH. Afterwards, the

proteins were redissolved in 2 mL Gua-HCl (6 M with 0.5 M phosphoric

acid, pH 2.5). The absorbance was measured at 370 nm using a UV–Vis

Spectrophotometer (Helios Gamma, Thermo Spectronic, Cambridge,

UK). Protein carbonyls were calculated using the molar attenuation

coefficient of 2.2 ⋅ 10

4

1

and were expressed as nanomoles of

carbonyls per milligram protein. All results were corrected by the pro-

tein concentration which was determined by measuring the absorbance

of the precipitated and resuspended sample at 278 nm and by use of the

molar extinction coefficient of 17,600 M

1

The presence of dityrosine (DiTyr) and N-formyl-kynurenine (NFK)

in the protein solution at pH 7 with a protein concentration of 1 mg/mL

were analyzed in quartz cuvettes using a fluorescence spectrophotom-

eter (Varian Cary Eclipse, Varian, Darmstadt, Germany) as described by

Scheidegger et al. (2010). For DiTyr and NFK measurements, an exci-

tation wavelength of 325 nm and emission wavelengths of 410 and 435

nm were used, respectively.

2.8. Surface activity and emulsifying properties

2.8.1. Adsorption behavior and interfacial rheology

To determine the adsorption behavior of the different BLG variants

as well as their interfacial rheology, drop tensiometry with an oil/water

(O/W) system was conducted.

Medium-chain triacylglyceride oil (MCT-oil) was used as the organic

phase. To remove free fatty acids and other interfacial active com-

pounds, MCT-oil was treated with Florisil as previously described

(Schestkowa et al., 2019). The adsorption behavior of BLG at the O/W

J.K. Keppler et al.

Food Hydrocolloids 110 (2021) 106132

interface was carried out with the pendant drop method using the profile

analysis tensiometer (PAT1M, Sinterface Technologies, Berlin, Ger-

many) with a two-fluid needle system as described by Schestkowa et al.

(2019). Shortly, an ultrapure water droplet with a profile area of 50 mm

was dosed with the outer needle into the MCT-oil in a quartz-glass

cuvette. Then, 2

L of the water droplet was exchanged with 2

L of

20 mg/mL (2 wt%) BLG solution (prepared in ultrapure water) through

the inner needle (resulting BLG concentration in the pendant drop was

0.1 wt%). The interfacial tension analysis of the single drop was per-

formed for 0.5 h at 22 �C by extracting the droplet shape recorded with a

high-speed camera. The migration of the BLG through the droplet is

characterized by the lag-time and was calculated in accordance with

Schestkowa et al. (2019).

After the drop aging of 0.5 h, a subsequent amplitude sweep ranging

from 0.1 to 7% deformation was performed at a frequency of 0.01 Hz as

described by Tamm and Drusch (2017). The elastic (storage) modulus E

and viscous (loss) modulus E” were derived from E*, which was calcu-

lated from the oscillation cycles. For E’ >E’’, the interfacial film is

predominantly elastic, while for E’ <E”, the interfacial film is pre-

dominantly viscous. The property of the interfacial film is a character-

istic parameter for the interfacial stabilization of emulsions.

2.8.2. Emulsifying properties

A coarse oil-in-water emulsion was prepared by dispersing 5% (w/w)

purified MCT-oil (dispersed phase) in a 1 mg/mL (0.1 wt% BLG solution

(continuous phase), prepared with ultrapure water) using a rotor-stator

homogenizer (Ultra Turrax T25 basic, 500 W power output, S25 KD-25 F

dispersion tool, IKA, Staufen, Germany) at 6500 min

1

for 15 s. Then,

the coarse emulsions were pushed through a silicate membrane (mem-

brane diameter 10 mm, membrane height 2.0 �0.2 mm median pore

diameter 10–16

m) to produce a fine emulsion. A new membrane was

used for each emulsification. The oil droplet size distribution (D10, D50,

D90, cumulative distribution function Q

) of the coarse and fine emul-

sion was measured with a laser scattered light spectrometer (Horiba LA-

950, Retsch Technology, Haan, Germany; transmission range: 80–95%

(R) and 70–90% (B), refractive index: 1.46). Each sample was analyzed

in analytical triplets, and the mean average of the analytical triplet was

used as one technical measurement. Individual aliquots of 10 mL of the

fine emulsions were transferred into graduated test tubes to check the

emulsion stability after 1, 2, 3, and 144 h (7 d) visually.

2.9. Statistics

If not stated otherwise, all sample solutions were prepared in tripli-

cate. Statistical significance was determined by calculating a one-way

analysis of variance (ANOVA) and by using Tukey’s multiple compari-

sons test as a post-hoc test. A significance level of 5% was assumed. The

tests were performed using GraphPad Prism (version 6.07, GraphPad

Software, San Diego, USA) or Statgraphics (Statgraphics Technologies

Inc., version 5.1, the Plains, Virginia, USA).

3. Results

3.1. Production and purification of recombinant beta-lactoglobulin B

(BLG B)

Within this study, two different bovine beta-lactoglobulin isoform B

proteins, rBLG B with an additional methionine residue at the N-termi-

nus (PDB ID: 5K06) and sBLG B (PDB ID: 6QI6) with a substituted N-

terminus L1A/I2S as described by Loch et al. (2016) were recombinantly

produced and purified (Table 1 and Fig. 1 and Fig. S1).

The purification process developed here using anion exchange

chromatography followed by an ammonium sulfate precipitation of

further E. coli host proteins and subsequent desalting resulted in >90%

purity of the corresponding protein (Fig. S1). During this newly devel-

oped procedure, the proteins were kept under innocuous conditions to

avoid stressful pH values or long times of dialysis, which were described

by Ponniah et al. (2010) before. With these newly developed production

and purification protocol including the addition of glucose to the growth

medium combined with an improved purification procedure applying

salting-out instead of size exclusion chromatography, the total yield of

purified BLG B was significantly increased from up to 40 mg described

previously (Loch et al., 2016) to 160 mg from 1 L of bacterial culture

maintaining a high purity.

3.2. Properties of the BLG variants

Because of the changed production process, the proteins themselves

were characterized in detail (e.g., N-terminal sequencing, mass spec-

trometry, thermal stability) to compare them with previously reported

analyses by Loch et al. (2016). A number of additional analyses have

been carried out that have not yet been done before with recombinant

BLG (e.g. MD modeling, protein oxidation, dissociation constants by

analytical ultracentrifugation).

3.2.1. Primary to tertiary structural properties of BLG variants

In the following, the two recombinant BLG B variants were compared

to BLG B and BLG AB, which were isolated from bovine milk. All proteins

used in this study had a purity higher than 90%. N-terminal sequencing

proofed the differences in the N-termini of the recombinant proteins and

the commercial BLG B. While rBLG B starts with methionine followed by

leucine, isoleucine, and valine (Table 1, Fig. 1, Fig. S2), the starting

methionine of sBLG B was missing due to cleavage during the recom-

binant production process as described before (Loch et al., 2016).

Because of the introduced mutations supporting the N-terminal methi-

onine cleavage in vivo, the first two amino acids in the mature sBLG B

were found to be alanine and serine. No mixture of proteins with

different N-termini could be found (Fig. S2).

Protein measurements by Qq TOF MS confirmed that covalent

modifications of the proteins did not occur during production and pu-

rification processes. The deconvoluted signals at charge state þ12 led to

an expected molecular mass of 18,288.9 Da for the commercial BLG B

sample, while sBLG B gave a 68 Da lower mass of 18,220.9 Da and rBLG

B a higher mass of additional 131 Da (Table 1). The amount of accessible

sulfhydryl groups (SH-groups) of the BLG variants can be used as a

Fig. 1. Superposition (C

) of lactoglobulin crystal structures: BLG B (PDB ID:

3NPO), rBLG B (PDB ID: 5K06) and sBLG B (PDB ID: 6QI6). The overall

structure of all compared variants is almost the same but N-terminal part of the

variant with additional N-terminal methionine (cyan) has different conforma-

tion than observed in natural protein isoform B (green) or a variant with N-

terminal mutations L1A/I2S (pink). (For interpretation of the references to

colour in this figure legend, the reader is referred to the Web version of

this article.)

J.K. Keppler et al.

Food Hydrocolloids 110 (2021) 106132

measure of nativity or correct folding. With respect to the number of

reactive thiol groups (determined in the RSH test), the present results

show that both recombinant variants behave within the range of the

bovine variants AB (0.43 mol/mol) and B (0.35 mol/mol), although

rBLG B had a significantly higher thiol group accessibility than sBLG B

(0.45 mol/mol and 0.38 mol/mol, respectively).

The amount of amino groups (NH groups, Table 1) of BLG variants

gives an indication of covalent modifications (decrease of NH groups) or

hydrolysis (increase of NH). Between 17 and 19.6 OPA (o-phtalalde-

hyde) amino groups could be observed for all BLG variants, with bovine

BLG AB having the highest number of 19.6 detectable groups.

A significant difference in the measured isoelectric point (IEP) from

4.7 to 4.9 is evident between BLG B and BLG AB (Fig. S3). In addition to

that, the recombinant BLG variants had a higher IEP than both reference

proteins with 5.4, which could be caused by different ions present or

some impurities because the theoretical IEP based on the unfolded

amino acid chain for BLG B, sBLG B, and rBLG B are similar (IEP at pH

4.83). On the other hand, the IEP of unfolded BLG is generally lower

than the IEP measured for native protein, with the exception of BLG B

(Table 1). Such a difference between the IEP of native and denatured

protein is only evident in some proteins due to specific amino acid

composition and folding and caused by intramolecular amino acid in-

teractions and lower solvent accessibility of buried amino acid groups in

the native protein which can cause local charge differences (Ui, 1971).

The BLG acid solubility is a rough measure of its nativity and correct

folding and is based on the lower IEP of unfolded BLG compared to

native BLG (Table 1). Thus, denatured BLG precipitates at pH 4.6, while

native BLG remains in solution. The BLG acid solubility was in the range

of >96% for all proteins, which can be considered native. Again, the

commercial protein BLG B deviated significantly from all other proteins

with only 88% solubility (Table 1).

Molecular dynamic (MD) analysis of the three BLG B variants was

performed to analyze structural differences in detail. Root mean squared

deviations (RMSD) of monomers, whether in dimeric form or pulled

apart in monomeric form, was slightly higher for rBLG B compared to

combined sBLG B and BLG B: 0.36 �0.068 nm vs. 0.29 �0.022 nm; p <

0.02. The higher fluctuation for rBLG B is presumably dominated by the

elongated N-terminal that, in all variants, remains only loosely attached

to the remainder of the protein, whether dimeric or monomeric.

Correspondingly, the radius of gyration was at 1.544 �0.016 nm for

rBLG B vs. 1.524 �0.0088 nm for combined sBLG B and BLG B with an

overall marginally significant difference (p <0.08).

This further corresponded to a different solvent accessible surface

area (SASA). The SASA of single monomers is larger for rBLG B than for

combined BLG B and sBLG B (183.0 �1.75 nm

vs. 178.7 �1.01 nm

; p

<10

3

). In the dimeric state (calculated per monomer), the difference in

the SASA remained, however on a lower level (168.9 �1.08 nm

vs.

165.6 �1.01 nm

; p <2⋅10

3

) while commercial and sBLG B cannot be

distinguished based on SASA. Pulling forces needed to monomerize di-

mers were indistinguishable for all variants. The same held true for

monomer-monomer binding energy. The number of stabilizing intra-

molecular hydrogen bonds has been found to be indistinguishable for all

BLG variants: A larger number of bonds was identified for the dimeric

state (calculated as the intramolecular H-bonds per monomer) of all BLG

variants together (i.e., mean of 232 �2.4) vs. a mean value of 224 �1.9

for all BLG variants in the monomeric state, but no differences were

found between BLG B, sBLG B or rBLG B.

Fourier transform infrared (FTIR) spectroscopy was conducted to

identify differences in the secondary and tertiary structural levels be-

tween the different BLG variants. The typical conformation of natively

folded BLG was evident for BLG B, i.e., the dominant intensity in the

1630 cm

1

band (Fig. 2A), indicating intramolecular beta-sheets, a less

dominant alpha-helix at 1655–1658 cm

1

and a second beta-sheet band

at 1688 cm

1

(Keppler et al., 2017; Keppler, Martin, Garamus, &

Schwarz, 2015; Panick, Malessa, & Winter 1999). The BLG AB likewise

showed a typical spectrum in the ATR-FTIR analysis with minor

variances in the turns and disordered elements region compared to BLG

B. There was also a minor band located at 1618 cm

1

, caused by

intermolecular beta-sheets. The two recombinant variants sBLG B and

rBLG B, however, exhibited a good resemblance to the BLG B spectrum,

since the intensity difference spectra gave no strong deviations (<0.001

A.U.) (Fig. 2B).

The intrinsic tryptophan (Trp) fluorescence of BLG (Fig. 2C) is a

sensitive measure of the accessibility of the aromatic amino acid Trp to

the solvent. All BLG variants were again very similar in their

Fig. 2. A) ATR-FTIR second derivative amide I spectra of beta-lactoglobulin

(BLG) variants. Waveband frequency regions for specific conformations (beta-

sheets, helix, disordered) are indicated. B) FTIR amide I difference spectra

between BLG B minus sBLG B or BLG B minus rBLG B. C) Intrinsic tryptophan

fluorescence spectra between 300 and 400 nm emission for different

BLG variants.

J.K. Keppler et al.

Food Hydrocolloids 110 (2021) 106132

fluorescence intensity (approximately 1.8–2.3 A.U.) as well as emission

wavelength maximum (approx. 350 nm wavelength). A slightly higher

fluorescence intensity was observed for the BLG AB.

3.2.2. Quaternary structure

AUC can be used to analyze the sedimentation profile of a sample

during centrifugation in real-time and it is thus possible to extract mo-

lecular size distributions in detail. The analysis of the monomer-dimer

dissociation constant K

of protein-protein interactions as a function

of the pH value by AUC was first conducted with two samples (BLG AB

and sBLG B) to determine the difference span (Fig. 3A). As can be seen,

the K

was almost similar for both tested variants. K

decreased at pH 5

because of aggregation effects at the isoelectric point (IEP), while it

increased towards more extreme pH values for both variants. Therefore,

the remaining samples were measured only at one fixed protein con-

centration and at pH 7 and the monomer-dimer equilibrium was

assessed based on these results.

The AUC results confirmed the predominant presence of dimers (36

kDa) for dialyzed BLG AB, sBLG B, and rBLG B at pH 7 in 10 mM NaCl, as

well as some monomers (18.5 kDa) and multimers >50 kDa (Fig. 3B).

The small differences in the molecular weight of the different variants

(Table 1) did not affect sedimentation and diffusional properties within

sedimentation velocity (SV) AUC experiments. Moreover, the monomer-

dimer equilibrium (Fig. 3A) was not different for the different BLG

variants tested, hence the quaternary structure of the recombinant

proteins has not been affected by the N-terminal modifications. Like-

wise, the hydrodynamic diameter measured in PBS using dynamic light

scattering (DLS) gave the size of dimers between 5 and 6 nm, which was

in accordance with results from AUC (Fig. 3, Table 2).

Zeta potential measurements (Table 2) showed minor differences

between the reference proteins BLG AB and B (both 18.6 mV at pH 7)

and the two recombinant variants (both - 21 mV).

3.2.3. Physicochemical properties

Different experiments were conducted to characterize the effect of

the amino acid addition or substitution at the N-termini of the recom-

binant BLG B variants on their physicochemical properties compared to

commercial BLG B and to BLG AB.

The denaturation onset temperature (T

onset) of the natural bovine

variants is an indication for the protein stability and its propensity to

unfold, but also an important measure for its thermal stability during

processing. T

onset, as measured by differential calorimetry (DSC)

(Fig. S4), was similar for both reference proteins with approximately 73

�C and increased significantly from BLG B to sBLG B by 1.7 K and from

for BLG B and rBLG by 3.4 K. Protein denaturation was completed at

higher temperatures between 77 �C for both reference proteins BLG B

and BLG AB. The sBLG B showed a T

less than 1 K higher, however,

rBLG B differed by 3.2 K from BLG B.

Chromatographic separations according to protein hydrophobicity is

shown in Fig. 4. The hydrophobicity of a protein is an important mea-

sure for its surface activity. The polarity of the protein decreases with

increasing retention time (RT). While the BLG B eluted at 18.4 min, rBLG

B eluted at 19 min and sBLG B at 17.8 min. The BLG AB reference

confirmed that BLG B eluted at 18.4 min, while BLG A eluted at 19.5

min. When the proteins are completely unfolded (Fig. 4B) the hydro-

phobicity of initially hidden amino acids can be taken into account with

this method. As can be seen, a similar elution order, albeit with a longer

elution time difference is now evident. Due to the presence of Gua-HCL,

however, all proteins elute now between 12 and 14 min (Fig. 4B) instead

of 17 and 20 min (Fig. 4A).

3.3. Oxidation state

Protein oxidation that occurs during purification, processing, and

storage of proteins, results in addition to post-translational modification

in the organism to chemical modification of the BLG. Typical protein

oxidation markers are protein-bound carbonyls detected by 2,4-dinitro-

phenylhydrazine (DNPH) derivatization as well as simple fluorescence

measurements at Ex325nm/Em415nm and Ex325nm/Em435nm, which

could be indicative for dityrosine oxidation (DiTyr) as a consequence of

tyrosine oxidation and N-formylkynurenine formatione (NFK) as one of

several tryptophan oxidation markers (Fig. 5) (Scheidegger et al., 2010).

The content of protein-bound carbonyls (Fig. 5A), however, is a

reliable marker to assess the general oxidation state of proteins. All

samples showed an overall low oxidation with respect to protein

carbonyl (<1 nmol per mg of protein) or fluorescence formation (<30

Int A.U.). As NFK (Fig. 5B) and DiTyr (Fig. 5C) fluorescences showed

overlap, the results could not clearly distinguish between Tyr or Trp

oxidation. The results confirmed, that the recombinant production and

isolation procedure had no detrimental effect on the oxidative state of

the protein.

Fig. 3. A) Chemical equilibrium constant in terms of the dimer dissociation

constant (K

) derived from the monomer-dimer equilibrium of sBLG B and BLG

AB as determined from SV AUC experiments at six different concentrations

(0.25–1.5 g/L) in water set to pH values of 3, 5, 7 and 9. B) Sedimentation

coefficient distribution as well as estimated molar masses from AUC data

analysis for BLG B, sBLG B and rBLG B in water at pH 7 measured at a con-

centration of 1 g/L and rotor speed of 50,000 min

1

Table 2

Size and zeta-potential of all BLG variants used in this study.

BLG AB BLG B rBLG B sBLG B

Zeta Potential in 10 mM NaCl

(pH 7) [mV]

18.6 �

1.2

18.7 �

1.5

21 �

0.6

21 �

1.6

Hydrodynamic diameter in

PBS (pH 7) [nm]

5.4 �0.5

5.2 �0.3

5.4 �

0.3

6.0 �

0.4

and

: significantly different values (p <0.05) are indicated with different

letters in a row.

J.K. Keppler et al.

Food Hydrocolloids 110 (2021) 106132

3.4. Surface activity measurements

3.4.1. Interfacial tension

The dynamic interfacial tension curves (Fig. S5) give an indication of

the propensity of a protein to unfold and align at a liquid interface and

thus help to draw conclusions about its emulsifying properties. The

curves were used to calculate the lag-time (i.e., time until the first pro-

teins adsorb and unfold at an interface), interfacial adsorption rate (i.e.,

the velocity at which the interface is rearranged and occupied with

proteins) and the equilibrium interfacial tension for stable systems (i.e.,

the ability to stabilize the interfacial layer) (Table 4). The lag-times were

similar with approximately 10 s for all variants except for the BLG AB

mixture with 28 s.

In contrast, the adsorption rate of BLG AB was the highest (1.05 mN

1

) while BLG B exhibited the lowest rate with 0.12 mN m

1

This could also be seen optically from the decrease of the interfacial

tension (Fig. S6). The long-time equilibrium interfacial tension of the

BLG B was also significantly higher (~20 mN m

1

) compared to the

other BLG variants (~15 mN m

1

). When comparing only the re-

combinant variants, they showed no significant differences in their

surface-active properties. They could be ranked between the BLG B and

BLG AB samples.

Amplitude sweeps were used to investigate the physical properties of

the protein layers at the O/W interface (Fig. S6). The dilatational elastic

(E’) and viscous (E”) moduli were plotted as a function of the applied

deformation of 0.8, 3.5, and 7%. All BLG films at the oil-water (O/W)

interface exhibited viscoelastic properties (E’>E”) (Fig. 6) with minor

variations. The sBLG B film showed significantly lower elastic modulus

E’, which correlates to less elastic properties in the film compared to

bovine BLG B and AB, while the rBLG B was in between (sBLG B <rBLG

B <BLG B).

3.4.2. Emulsion oil droplet size

The mean emulsion oil droplet size of all proteins in the premix as

well as in the final emulsion was not significantly different between the

recombinant and the bovine variants (Fig. 7), except for the D90 sizes of

the BLG B and rBLG B stabilized emulsion which were slightly larger

than the D90 sizes of BLG AB and sBLG B. The fine emulsions produced

by premix membrane-emulsification, where the resulting droplet size is

determined by the used membrane pore size (10–16

m), showed mean

oil droplet sizes of approximately 13

m size. In addition, also the

creaming of the BLG stabilized emulsions (fine emulsions stored for 7

days) showed no differences and no separation of the dispersed phase

(data not shown).

4. Discussion

The experiments were conducted to investigate how the single amino

acid changes in the sequence of BLG variants affect the protein structure,

physicochemical properties and finally, the emulsifying properties.

4.1. Structural properties

The BLG B variants differ in their N-terminal region, which affects

the calculated and measured molecular weight. The lower mass of the

sBLG B mutant compared to BLG B can be explained by the amino acid

exchanges at positions 1 and 2 (Table 1, Fig. 1), which confirms a

theoretical mass difference of 68 Da. In contrast, the additional

methionine at the N-terminus of rBLG B adds 131 Da to the protein mass.

Similar findings for rBLG B have been described in the literature (Loch

et al., 2016).

Approximately 16 amino groups (i.e., 15 epsilon-lysines and the N-

terminal alpha amino group) can ideally be measured within native BLG

A or B (Chevalier, Chobert, Popineau, Nicolas, & Haertl�

e, 2001), which

is roughly in the range of the present results (17–20 NH groups) and also

agrees with literature reports of 18–19 free amino groups per mol BLG

AB (Keppler et al., 2014; Rade-Kukic, Schmitt, & Rawel, 2011). Thus, no

modifications on the lysine residues were found for the present recom-

binant BLG variants which was confirmed by the MS data (Table 1). A

significantly higher number of amino groups would have indicated hy-

drolysis by e.g., oxidation (Church, Swaisgood, Porter, & Catignani,

1983; Liu & Xiong, 2000) while a significantly lower number would

indicate blocking or reduced accessibility of the amino group for the

OPA reagent, as caused by e.g. lactozylation or aggregation (Kerkaert

et al., 2011; Losito, Stringano, Carulli, & Palmisano, 2010; Norwood

et al., 2016).

With respect to the amount of accessible thiol (SH) groups, there

seem to be only minor structural differences. The presented results are

comparable to the 0.2–0.3 mol SH groups per mol protein which have

been reported for a correctly folded BLG AB in the native state (Kehoe

et al., 2007; Kehoe, Remondetto, Subirade, Morris, & Brodkorb, 2008;

Keppler et al., 2014), where the free thiol group of Cys121 is mostly

inaccessible. A maximum of 1 mol SH group per mol protein can be

measured when BLG would be completely unfolded. The significantly

higher amount of accessible SH groups of rBLG B could indicate minor

Table 3

Characterization of all BLG variants used in this study.

BLG AB BLG B rBLG B sBLG B

onset [�C] 73.0 �0.1

73.3 �0.1

76.7 �0.1

75.0 �0.1

[�C] 77.5 �0.1

77.3 �0.1

80.5 �0.1

78.2 �0.1

, denaturation temperature;

and

: significantly different values (p <0.05)

are indicated with different letters in a row.

Fig. 4. Detail of RP-HPLC chromatograms of different BLG variants: A) Native monomers and B) unfolded monomers. The BLG AB reference (dotted black line)

shows the presence of both isoforms A and B denoted as A and B in the chromatogram, sB and rB indicate recombinant variants sBLG B and rBLG B, respectively.

J.K. Keppler et al.

Food Hydrocolloids 110 (2021) 106132

folding differences, higher structural flexibility of the protein , or

thiol-containing impurities. Since the FTIR results show no significant

differences, the secondary structure seems to be mostly unaffected: The

observed conformation of all BLG variants (Fig. 2A and B) is mostly

comparable and in line with other FTIR analyses for native BLG B or BLG

AB (Keppler et al., 2015; Panick et al., 1999). In addition, the Trp

fluorescence analyses (Fig. 2C) also indicate no significant intensity

increases or wavelength shifts which would have been indicative of

conformational differences between the proteins for example with

respect to solvent accessibility (Eftink & Ghiron, 1976; Heyn et al.,

2019). The presented results confirm that both recombinant proteins are

indeed correctly folded, as stated previously by Loch et al. (2016)

(Table 1, Fig. 1). In addition, all samples showed an overall low oxida-

tion with respect to protein carbonyl (<1 nmol/mg protein) or fluores-

cence formation (<30 Int A.U.) (Fig. 5). Similar low values were found

for whey protein powders (Keppler, Heyn, Meissner, Schrader, &

Schwarz, 2019; Scheidegger et al., 2010; Semagoto et al., 2014).

Hence, the presented results confirm that the recombinant produc-

tion in the bacterial host E. coli and isolation procedure have no detri-

mental effect on the nativity, PTMs, and oxidative state of the produced

BLG variants.

Fig. 5. Protein oxidation markers: A) protein-bound carbonyls, B) N-for-

mylkynurenine (NFK) fluorescence and C) dityrosine (DiTyr) fluorescence) for

different BLG variants. All measurements were conducted in triplicate and are

listed as mean �standard deviation. Different letters indicate statistically sig-

nificant differences (p <0.05).

Table 4

Adsorption parameters (lag-time, interfacial adsorption rate, and interfacial

tension) for BLG AB, BLG B, rBLG B, and sBLG B.

BLG AB BLG B rBLG B sBLG B

lag-time [s] 27.9 �

11.4

12.3 �

3.4

a,b

6.8 �1.4

9.8 �4.7

Interfacial adsorption rate

[mN m

1

]

1.1 �

0.7

0.1 �

0.0

0.5 �

0.3

a,b

0.9 �

0.4

a,b

Interfacial tension for t→∞

[mN m

1

]

15.8 �

0.7

20.2 �

0.3

14.9 �

1.0

16.7 �

1.1

and

: significantly different values (p <0.05) are indicated with small letters

for a row.

Fig. 6. A) Storage modulus E’ and B) loss modulus E’’ of the interfacial film of

BLG AB, BLG B, rBLG B, and sBLG B at the deformation of the interfacial area of

0.8, 3.5 and 7.0%. Different letters (a–e) show significant differences between

the values (p <0.05).

Fig. 7. Oil droplet size D10 (white), D50 (light grey), D90 (black) for the coarse

(filled bars) and the fine emulsion (dotted bars) stabilized with BLG AB, BLG B,

rBLG B or sBLG B derived by premix membrane emulsification. Different letters

(a–d) show significant differences between the values (p <0.05).

J.K. Keppler et al.

Food Hydrocolloids 110 (2021) 106132

Overall, the MD simulations confirm that the BLG variants showed

high structural similarities (Table 1), with sBLG B performing most

similar to the commercial BLG B, while rBLG B exhibits slightly reduced

structure stability as indicated by the significantly higher root mean

squared deviation (0.36 nm vs. 0.29 nm), the higher radius of gyration

(1.54 nm vs. 1.52 nm) and the higher solvent accessibility (183 nm

vs.

178 nm

The determination of the quaternary structure of the BLG variants by

AUC as well as by DLS also confirms a similar monomer/dimer behavior

for the recombinant variants as well as similar K

for BLG AB and re-

combinant BLG Bs (Fig. 3A and B, Table 2). Correspondingly, MD sim-

ulations at neutral pH resulted in the same monomer/monomer binding

energy for all variants. The results are in agreement with the literature

on the monomer/dimer behavior of BLG B and rBLG B (Mercadante

et al., 2012; Kristiansen, Otte, Ipsen, & Qvist, 1998). BLG was found to

be predominantly monomeric near pH 2 and 9, while the dimer form

prevails at pH 7 (Renard, Lefebvre, Griffin, & Griffin, 1998; Verheul,

Pedersen, Roefs, & Kruif, 1999).

4.2. Physicochemical properties

So far, the results confirmed that neither the protein production

system nor the isolation process affects the protein’s structural proper-

ties (i.e., high purity, native conformation (Fig. 2), neither oxidative

(Fig. 5) nor glycozylation based PTM (Table 1)). In addition, all BLG

variants showed similar folding and similar monomer/dimer equilib-

rium (Figs. 1, Figure 2, and Fig. 3, Table 2). Now, denaturation tem-

perature at pH 7, isoelectric point, zeta potential at pH 7, acid solubility,

and hydrophobicity were analyzed and compared (Tables 1–3).

As a first observation, the rBLG B displayed incomplete resolubili-

zation after lyophilization resulting in some undissolved protein ag-

gregates (<7%) which was not observed for the other variants. The

aggregates were removed by centrifugation or filtration (0.45

membrane) to allow spectroscopic or scattering analyses (e.g., protein

oxidation, OPA, RSH, DLS, AUC). The reason for the limited resolubili-

zation, which only concerns rBLG B, is unclear.

A significant difference can be observed for both recombinant vari-

ants compared to the other BLG variants with respect to the thermal

stability (Table 3). While BLG B and BLG AB exhibit denaturation tem-

peratures of approximately 77 �C in water (pH 7), sBLG B denatures at

~78 �C, and rBLG B at ~80 �C. In other studies, denaturation temper-

atures of BLG AB determined at neutral pH and low ionic strength varied

between 69.2 and 80.1 �C (Tolkach & Kulozik, 2007; Busti, Gatti, &

Delorenzi, 2005). The results of the present study are consistent with the

reported denaturation temperature range. The reported bandwidth of

variation (10 K) of these measurements are caused by differences in

protein purity or analysis methodology. In contrast to the present

finding, CD analyses gave evidence of a slightly lower thermal stability

for sBLG B than BLG B (Loch et al., 2016). Also these differences could be

caused by using different purification strategies of sBLG B, different

analysis methods and conditions (e.g. protein concentration, salt con-

centrations). Nevertheless, an effect of the modified N-terminus on the

denaturation temperature cannot be excluded, since a modified N-ter-

minus can have a stabilizing or even destabilizing effect on other pro-

teins (Chaudhuri, Horii, & Yoda, 1999) Schultz et al., 1992). However,

also the native genetic variants BLG A, B, and C (which differ by 1 or 2

amino acids) are found to diverge in their T

each by 5 K in phosphate

buffer (pH 7) (Keppler, S€

onnichsen, Lorenzen, & Schwarz, 2014) or

other solvents (Imafidon, Ng-Kwai-Hang, Harwalkar, & Ma, 1991;

McLean, Graham, Ponzoni, & McKenzie, 1987). Thus, the observed de-

viations of the recombinant proteins are within the natural variances of

BLG.

In general, an IEP of 5.1–5.2 is reported for BLG in the literature (Yan

et al., 2013) which is roughly in the range of our samples (IEP 4.9–5.4).

However, the IEP of commercial BLG B sample with 4.7 is lower than

expected (i.e., 5.23 for native BLG B). This deviation could indicate

denaturation, but this was not observed in FTIR and Trp fluorescence

(Fig. 2). In addition, the different IEP of the commercial BLG B was also

not reflected in the results of the quarternary structure analyses by AUC

(Fig. 3) or DLS (Table 2). Low ionic strength can lead to a loss of solu-

bility of globular proteins and the presence of multivalent (bound) an-

ions can extend the iso-ionic range to a pH of 4.6. Different ions or ion

concentrations in the commercial BLG B sample (obtained from Sigma

Aldrich) compared to those that have been isolated and dialyzed by our

groups (BLG AB, sBLG B, and rBLG B) could be an explanation for this

(de Wit & Kessel, 1996). Because of the high similarity of the IEP of the

other BLG variants, the acid solubility (i.e., solubility at pH 4.6) can still

be used as a measure of protein nativity in these cases. This analysis is

strongly dependent on the IEP shift between native and denatured BLG

(Table 1), thus denatured BLG precipitates at pH 4.6 while the native

protein remains in solution (Pizzano, Manzo, Nicolai, & Addeo, 2012;

Toro-Sierra et al., 2013). For the BLG AB and recombinant B variants,

the high acid solubility can be translated to a high nativity, which is also

in line with FTIR (Fig. 2A), Trp fluorescence (Fig. 2C) and the number of

accessible SH groups (Table 1). As expected, the low IEP observed in

BLG B also correlates with a reduced acid solubility of 88% and it can be

hypothesized that this result is likely rather connected to the solubility

issues discussed before and not to its state of denaturation because FTIR

and Trp fluorescence indicate high nativity.

Finally, the protein hydrophobicity was analyzed using an RP-HPLC,

which is independent of the ionic strength in the samples. The observed

difference in elution time indicates that BLG A is more hydrophobic than

BLG B which has already been reported by Keppler et al. (2017) or

Ruprichova et al. (2014) using a similar chromatographic separation

technique. Although, at neutral pH values, the overall net negative

charge for BLG A is higher than BLG B because of the negative charge of

the Asp at position 64 (Keppler, S€

onnichsen et al., 2014). Overall it is

difficult to forecast the hydrophobic behavior of proteins in chromato-

graphic separations based only on the amino acid sequence (Mahn,

Lienqueo, & Asenjo, 2004). It was shown, that the retention in RP-HPLC

that is based on hydrophobic interactions is strongly influenced by the

flexibility of proteins (To & Lenhoff, 2007). This explains the observed

behavior of BLG A and BLG B in RP-HPLC: BLG A interacts more strongly

with the chromatographic stationary phase than BLG B despite only two

changes in the amino acid composition (BLG A: Asp64, Ala118. BLG B:

Gly64, Val118). Based on the change in Position 118, which is located

inside the protein’s core, Qin et al. reported a higher flexibility for BLG A

(Qin, Bewley, Creamer, Baker, & Jameson, 1999) that may explain the

observed behavior of BLG variants A and B in RP-HPLC. A simpler

relationship between amino acid exchange and hydrophobicity can be

seen in the recombinant variants, probably because the N-terminal

amino acid exchange has less effect on the protein backbone flexibility

than a mutation in the core of the protein. The substitution of the more

polar Ser in sBLG B against the nonpolar Ile explains the lower hydro-

phobicity of sBLG B compared to BLG B. The rBLG B, on the other hand,

contains an additional nonpolar Met, which shifts the protein to a higher

residence time in RP-HPLC. However, further investigation on protein

structure and on the flexibility of the variants sBLG B and rBLG B is

necessary to explain the elution order.

4.3. Emulsifying properties

The present results show that the recombinant variants are struc-

turally very similar. The main differences between rBLG B and the other

BLG variants are the denaturation temperature and the resolubilization

behavior. Therefore, the impact on the functional level (i.e., interfacial

behavior and emulsifying properties) was investigated in the following.

The migration-driven interfacial activity of all BLG variants was

compared using pendant drop analysis. The dynamic interfacial tension

shows that the lag-time and equilibrium interfacial tension (Table 4) are

roughly in the range reported previously for whey protein isolate rich in

BLG in water (pH 7), adsorbed at a rapeseed O/W interface (Keppler

J.K. Keppler et al.

Related document tools

Why organizations use Identific for document trust, entry 28

Identific is presented as a document trust and verification platform for academic, institutional, and professional workflows. Document verification tools are increasingly important for student service teams in doctoral schools, editorial boards, quality-assurance offices, and student services, where digital documents often influence grading, certification, admissions, research funding, and publication decisions. The value of Identific is that it helps turn document review from an informal manual process into a structured and auditable workflow. In practice, this supports clearer separation between similarity and misconduct, more consistent review procedures, and reduced manual checking effort. Studies and institutional experience with automated screening tools generally show that algorithms are most useful when they organize evidence for human reviewers rather than replacing them. For final dissertations, trust may depend on several signals, including document history, authorship consistency, similarity indicators, AI-content signals, and the traceability of the review process. Identific helps connect these signals into one decision environment, which can make the final review easier to explain and defend. Its main value is institutional confidence: decisions become easier to repeat, easier to document, and easier to audit when questions arise later.

Identific

Food Hydrocolloids 110 (2021) 106132

et al., 2018; B€

ottcher, Keppler, & Drusch, 2017). Deviations are likely

caused by different methodologies and purities between the different

studies (BLG vs. whey protein isolate, different oil phases, and different

ions) (Beverung, Radke, & Blanch, 1999; Geerts, Nikiforidis, van der

Goot, & van der Padt; Keppler et al., 2018; Mackie, Husband, Holt, &

Wilde, 1999; Tenorio, Jong, Nikiforidis, Boom, & van der Goot, 2017).

All three measured surface properties (lag-time, adsorption rate and

equilibrium interfacial tension) are the result of different, complexly

interacting protein properties, so they can rarely be traced back to a

single effect and further analyses are needed to elucidate this. In the

following, the individual properties of the interfacial activity are dis-

cussed in more detail.

The lag-time correlates with the initial protein adsorption (short-

term effects). It is characterized by protein migration through the bulk

phase. In addition, the lag-time ends as soon as a protein adsorbs and

unfolds at an interface, thus a high interface denaturation kinetics of the

protein and a low surface charge correlate with a reduced lag-time

(Beverung et al., 1999; Keppler et al., 2018). The lag-times were similar

with approximately 10 s for all variants except for the BLG AB mixture

with 28 s. The similar lag-time for all BLG B variants can be explained by

their similar size (i.e., similar migration speed) and similar

zeta-potential (Table 2). The higher thermal stability of the recombinant

variants (Table 3) seems to have no overall impact on the lag-time. The

higher lag-time of the BLG AB might be explained by the higher net

negative surface charge of the BLG A at pH 7, causing less attraction to

hydrophobic interfaces than observed for BLG B. Although the overall

hydrophobicity of BLG A in HPLC is highest (Fig. 4, see discussion

above), the single negative charge on its surface can reduce the initial

adsorption compared to BLG B (Mackie et al., 1999).

The adsorption rate (Table 4) correlates to the mid-term structural

rearrangement of the protein at the interface. The overall hydropho-

bicity of the protein, molecular flexibility and conformational stability

were found to be of importance (Beverung et al., 1999; Keppler et al.,

2018). Again, all BLG B variants showed similar adsorption rates, while

the BLG AB differed. The high molecular flexibility of BLG A could be an

explanation for the overall faster unfolding and rearrangement of BLG

AB, despite its single negative charge on the surface. However, sBLG B

also showed a fast surface adsorption rate, but there was no evidence of a

high structural flexibility and/or a low overall hydrophobicity as it

showed the lowest stationary phase binding in HPLC (Fig. 4). Differences

in the adsorption rate can also be due to different electrostatic in-

teractions and densities of the BLG AB dimer compared to the BLG B

homodimers at pH 7 (Schestkowa et al., 2019).

The equilibrium interfacial tension correlates to the long-term ef-

fects, such as overall protein packing density at the interface, interface

rearrangement and network formation. The equilibrium interfacial

tension was significantly lower for BLG B compared to all other variants.

This could be due to a lower packing density of BLG B at the interface

(Keppler et al., 2018; Schestkowa et al., 2019), although the high elastic

properties of the BLG B surface film indicates a densely packed interface

or a well interconnected film (Fig. 6).

All BLG films at the O/W interface exhibited viscoelastic properties

(E’ >E”) with the sBLG B film showing less elastic properties in the film

compared to the commercial BLG B. This can indicate a less densely

packed interface (Bos & van Vliet, 2001; Engelhardt et al., 2013; Keppler

et al., 2017; Sagis & Scholten, 2014) and a lower mechanical stability,

but on the other hand, the recombinant variants showed no shear

dependent loss of storage modulus which confirms the stability against

some mechanical stress (Fig. 6).

Finally, all BLG variants resulted in emulsions with similar oil

droplet size distribution and creaming stability (Fig. 7). The droplet size

of the coarse emulsion stabilized with BLG AB (D50 ¼25

m) was

comparable to BLG B stabilized water/MCT-oil emulsions prepared with

a high-pressure homogenizer (10–20

m) (B€

ottcher et al., 2017). The

observed faster adsorption kinetics of the recombinant BLG variants

(Fig. 4) probably does not take effect in the investigated time range of

the emulsification process. The emulsion creaming stability is likewise

not different for the different proteins, probably due to the fact that the

low interfacial elasticity of the recombinant variants is compensated by

the high protein concentration (saturated interface) and will rather be

observable in less concentrated samples. Other homogenization pro-

cesses (e.g., ultrasonic), other continuous phases as well as other protein

concentrations could lead to differences in the adsorption behavior,

achieved droplet size and then displaying the observed differences in

adsorption behavior and interfacial stability.

In conclusion, in the present study similar emulsion properties were

found for all recombinant BLG variants as well as for bovine BLG isolated

from milk.

5. Conclusion

The presented preparation and isolation of recombinant BLG B var-

iants resulted in proteins with high nativity, purity, and no significant

PTM.

Based on the simulations and measurements shown, the sBLG B

seemed to exhibit higher equivalence with commercial bovine BLG B,

while the elongated N-terminal of the rBLG B led to deviations in radius

and structural stability. Nevertheless, all variants are structurally very

similar. Consequently, the emulsification properties are not affected by

the structural differences of the recombinant variants. Thus, these pro-

teins can be used from a functional point of view as bovine whey protein

substitutes with regard to emulsion production. As scientific/research

perspective, these recombinant proteins can be used as starting base for

targeted modifications of BLG for applications in food technology

beyond the varieties produced in this study.

Further tests regarding other homogenization methods, but also

other process steps such as foaming, drying, and gelling behavior are

still required in order to obtain a more holistic view of the performance

and the substantial equivalence of these proteins. These investigations

are relevant to assess the commercial and environmental sustainability

of this alternative concept of producing proteins.

Declaration of competing interest

None.

CRediT authorship contribution statement

Julia K. Keppler: Project administration, Funding acquisition,

Conceptualization, Resources, Writing - original draft, Visualization,

Supervision. Anja Heyse: Conceptualization, Investigation, Formal

analysis, Writing - original draft. Eva Scheidler: Investigation, Formal

analysis, Writing - original draft. Maximilian J. Uttinger: Investigation,

Formal analysis, Writing - original draft. Laura Fitzner: Investigation,

Formal analysis, Writing - original draft. Uwe Jandt: Formal analysis,

Writing - original draft, Funding acquisition. Timon R. Heyn: Investi-

gation, Formal analysis, Writing - original draft. Vanessa Lautenbach:

Investigation, Formal analysis, Writing - original draft. Joanna I. Loch:

Writing - review & editing, Visualization, Resources. Jonas Lohr:

Investigation, Writing - original draft. Helena Kieserling: Writing -

review & editing, Visualization. Gabriele Günther: Investigation.

Elena Kempf: Investigation. Jan-Hendrik Grosch: Investigation.

Krzysztof Lewi�

nski: Writing - review & editing, Resources. Dieter

Jahn: Writing - review & editing, Resources, Funding acquisition, Su-

pervision. Christian Lübbert: Investigation, Writing - original draft.

Wolfgang Peukert: Funding acquisition, Writing - review & editing,

Supervision, Resources. Ulrich Kulozik: Funding acquisition, Writing -

review & editing, Supervision, Resources. Stephan Drusch: Funding

acquisition, Writing - review & editing, Supervision, Resources. Rainer

Krull: Funding acquisition, Conceptualization, Writing - review &

editing, Supervision, Resources. Karin Schwarz: Funding acquisition,

Conceptualization, Writing - review & editing, Supervision, Resources.

J.K. Keppler et al.

Food Hydrocolloids 110 (2021) 106132

Rebekka Biedendieck: Project administration, Funding acquisition,

Conceptualization, Resources, Writing - original draft, Supervision.

Acknowledgments

The authors gratefully acknowledge the financial support provided

by the German Research Foundation (DFG) within the priority program,

SPP1934 “DiSPBiotech – Dispersity, structural and phase modifications of

proteins and biological agglomerates in biotechnological processes”. TU

Braunschweig would like to thank Beate Jaschok-Kentner, Helmholtz

Centre for Infection Research, Department of Molecular Structural

Biology, Braunschweig, Germany, for N-terminal protein sequencing.

Kiel University acknowledges the skillful help of Jesco Reimers, Division

of Food Technology, Kiel University, Germany, with protein fluores-

cence measurements. ES and UK acknowledge the professional technical

work of Claudia Hengst in conducting the HPLC analysis. UJ acknowl-

edges partial funding by BMBF (grant 031B0222).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.foodhyd.2020.106132.

References

Batt, C. A., Rabson, L. D., Wong, D. W., & Kinsella, J. E. (1990). Expression of

recombinant bovine beta-lactoglobulin in Escherichia coli. Agricultural & Biological

Chemistry, 54(4), 949–955.

Beverung, C. J., Radke, C. J., & Blanch, H. W. (1999). Protein adsorption at the oil/water

interface: Characterization of adsorption kinetics by dynamic interfacial tension

measurements. Biophysical Chemistry, 81(1), 59–80.

Bhat, Z. F., Kumar, S., & Fayaz, H. (2015). In vitro meat production: Challenges and

benefits over conventional meat production. Journal of Integrative Agriculture, 14(2),

241–248.

Bos, M. A., & van Vliet, T. (2001). Interfacial rheological properties of adsorbed protein

layers and surfactants: A review. Advances in Colloid and Interface Science, 91(3),

437–471.

B€

ottcher, S., Keppler, J. K., & Drusch, S. (2017). Mixtures of Quillaja saponin and beta-

lactoglobulin at the oil/water-interface: Adsorption, interfacial rheology and

emulsion properties. Colloids and Surfaces A: Physicochemical and Engineering Aspects,

518, 46–56.

Brown, P. H., & Schuck, P. (2006). Macromolecular size-and-shape distributions by

sedimentation velocity analytical ultracentrifugation. Biophysical Journal, 90(12),

4651–4661.

Busti, P., Gatti, C. A., & Delorenzi, N. J. (2005). Thermal unfolding of bovine

β-lactoglobulin studied by UV spectroscopy and fluorescence quenching. Food

Research International, 38(5), 543–550.

Chaudhuri, TK, Horii, K, Yoda, T, et al. (1999). Effect of the extra n-terminal methionine

residue on the stability and folding of recombinant alpha-lactalbumin expressed in

Escherichia coli. Journal of Molecular Biology, 285(3), 1179–1194. https://doi.org/

10.1006/jmbi.1998.2362.

Chevalier, F., Chobert, J.-M., Popineau, Y., Nicolas, M. G., & Haertl�

e, T. (2001).

Improvement of functional properties of β-lactoglobulin glycated through the

Maillard reaction is related to the nature of the sugar. International Dairy Journal, 11

(3), 145–152.

Cho, Y., Gu, W., Watkins, S., Lee, S. P., Kim, T. R., Brady, J. W., et al. (1994).

Thermostable variants of bovine beta-lactoglobulin. Protein Engineering, 7(2),

263–270.

Church, F. C., Swaisgood, H. E., Porter, D. H., & Catignani, G. L. (1983).

Spectrophotometric assay using o-phthaldialdehyde for determination of proteolysis

in milk and isolated milk Proteins. Journal of Dairy Science, 66(6), 1219–1227.

Dekkers, B. L., Boom, R. M., & van der Goot, A. J. (2018). Structuring processes for meat

analogues. Trends in Food Science & Technology, 81, 25–36.

Denton, H., Smith, M., Husi, H., Uhrin, D., Barlow, P. N., Batt, C. A., et al. (1998).

Isotopically labeled bovine β-lactoglobulin for NMR studies expressed in Pichia

pastoris. Protein Expression and Purification, 14(1), 97–103.

de Wit, J. N., & Kessel, T.v. (1996). Effects of ionic strength on the solubility of whey

protein products. A colloid chemical approach. Food Hydrocolloids, 10(2), 143–149.

Dombrowski, J., Gschwendtner, M., Saalfeld, D., & Kulozik, U. (2018). Salt-dependent

interaction behavior of β-Lactoglobulin molecules in relation to their surface and

foaming properties. Colloids and Surfaces A: Physicochemical and Engineering Aspects,

558, 455–462.

Dumpler, J., Wohlschl€

ager, H., & Kulozik, U. (2017). Dissociation and coagulation of

caseins and whey proteins in concentrated skim milk heated by direct steam

injection. Dairy Science & Technology, 96(6), 807–826.

Edman, P., & Begg, G. (1967). A protein sequenator. In C. Li�

ebecq (Ed.), European journal

of biochemistry (pp. 80–91). Berlin, Heidelberg: Springer Berlin Heidelberg.

Eftink, M. R., & Ghiron, C. A. (1976). Exposure of tryptophanyl residues in proteins.

Quantitative determination by fluorescence quenching studies. Biochemistry, 15(3),

672–680.

Ellman, G. L. (1959). Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics, 82

(1), 70–77.

Engelhardt, K., Lexis, M., Gochev, G., Konnerth, C., Miller, R., Willenbacher, N., et al.

(2013). pH effects on the molecular structure of β-lactoglobulin modified air–water

interfaces and its impact on foam rheology. Langmuir, 29(37), 11646–11655.

Geerts, M. E. J., Nikiforidis, C. V., van der Goot, A. J., & van der Padt, A. Protein nativity

explains emulsifying properties of aqueous extracted protein components from

yellow pea. Food Structure 14, 104-111.

Heyn, T. R., Garamus, V. M., Neumann, H. R., Uttinger, M. J., Guckeisen, T., Heuer, M.,

et al. (2019). Influence of the polydispersity of pH 2 and pH 3.5 beta-lactoglobulin

amyloid fibril solutions on analytical methods. European Polymer Journal, 120,

109211.

Hundschell, C. S., B€

ather, S., Drusch, S., & Wagemans, A. M. (2020). Osmometric and

viscometric study of levan, β-lactoglobulin and their mixtures. Food Hydrocolloids,

101, 105580.

Imafidon, G. I., Ng-Kwai-Hang, K. F., Harwalkar, V. R., & Ma, C.-Y. (1991). Differential

scanning calorimetric study of different genetic variants of β-lactoglobulin. Journal of

Dairy Science, 74(8), 2416–2422.

Jayat, D., Gaudin, J.-C., Chobert, J.-M., Burova, T. V., Holt, C., McNae, I., et al. (2004).

A recombinant C121S mutant of bovine β-lactoglobulin is more susceptible to peptic

digestion and to denaturation by reducing agents and heating. Biochemistry, 43(20),

6312–6321.

Kaminski, G. A., Friesner, R. A., Tirado-Rives, J., & Jorgensen, W. L. (2001). Evaluation

and reparametrization of the OPLS-AA force field for proteins via comparison with

accurate quantum chemical calculations on peptides. The Journal of Physical

Chemistry B, 105(28), 6474–6487.

Kayser, J. J., Arnold, P., Steffen-Heins, A., Schwarz, K., & Keppler, J. K. (2020).

Functional ethanol-induced fibrils: Influence of solvents and temperature on

amyloid-like aggregation of beta-lactoglobulin. Journal of Food Engineering, 270,

109764.

Kehoe, J. J., Brodkorb, A., Moll�

e, D., Yokoyama, E., Famelart, M.-H., Bouhallab, S., et al.

(2007). Determination of exposed sulfhydryl groups in heated b-lactoglobulin A

using IAEDANS and mass spectrometry. Journal of Agricultural and Food Chemistry, 55

(17), 7107–7113.

Kehoe, J. J., Remondetto, G. E., Subirade, M., Morris, E. R., & Brodkorb, A. (2008).

Tryptophan-mediated denaturation of β-lactoglobulin A by UV irradiation. Journal of

Agricultural and Food Chemistry, 56(12), 4720–4725.

Keppler, J. K., Koudelka, T., Palani, K., Stuhldreier, M. C., Temps, F., Tholey, A., et al.

(2014a). Characterization of the covalent binding of allyl isothiocyanate to

β-lactoglobulin by fluorescence quenching, equilibrium measurement, and mass

spectrometry. Journal of Biomolecular Structure & Dynamics, 32(7), 1103–1117.

Keppler, J., Martin, D., Garamus, V., Berton-Carabin, C., Nipoti, E., Coenye, T., et al.

(2017). Functionality of whey proteins covalently modified by allyl isothiocyanate.

Part 1 physicochemical and antibacterial properties of native and modified whey

proteins at pH 2 to 7. Food Hydrocolloids, 65, 130–143.

Keppler, J. K., Martin, D., Garamus, V. M., & Schwarz, K. (2015). Differences in binding

behavior of (-)-epigallocatechin gallate to beta-lactoglobulin heterodimers (AB)

compared to homodimers (A) and (B). Journal of Molecular Recognition, 28(11),

656–666.

Keppler, Heyn, Meissner, Schrader, & Schwarz. (2019). Protein oxidation during

temperature-induced amyloid aggregation of beta-lactoglobulin. Food Chemistry,

289, 223–231.

Keppler, J. K., S€

onnichsen, F. D., Lorenzen, P.-C., & Schwarz, K. (2014b). Differences in

heat stability and ligand binding among β-lactoglobulin genetic variants A, B and C

using 1H NMR and fluorescence quenching. Biochimica et Biophysica Acta (BBA) -

Proteins & Proteomics, 1844(6), 1083–1093.

Keppler, J. K., Steffen-Heins, A., Berton-Carabin, C. C., Ropers, M.-H., & Schwarz, K.

(2018). Functionality of whey proteins covalently modified by allyl isothiocyanate.

Part 2: Influence of the protein modification on the surface activity in an O/W

system. Food Hydrocolloids, 81, 286–299.

Kerkaert, B., Mestdagh, F., Cucu, T., Aedo, P. R., Ling, S. Y., & De Meulenaer, B. (2011).

Hypochlorous and peracetic acid induced oxidation of dairy proteins. Journal of

Agricultural and Food Chemistry, 59(3), 907–914.

Kim, T. R., Goto, Y., Hirota, N., Kuwata, K., Denton, H., Wu, S. Y., et al. (1997). High-

level expression of bovine beta-lactoglobulin in Pichia pastoris and characterization

of its physical properties. Protein Engineering, 10(11), 1339–1345.

Kristiansen, K. R., Otte, J., Ipsen, R., & Qvist, K. B. (1998). Large-scale preparation of

β-lactoglobulin A and B by ultrafiltration and ion-exchange chromatography.

International Dairy Journal, 8(2), 113–118.

Lam, R. S. H., & Nickerson, M. T. (2014). The effect of pH and heat pre-treatments on the

physicochemical and emulsifying properties of β-lactoglobulin. Food Biophysics, 9(1),

20–28.

Levine, R. L., Garland, D., Oliver, C. N., Amici, A., Climent, I., Lenz, A. G., et al. (1990).

Determination of carbonyl content in oxidatively modified proteins. Methods in

Enzymology, 186, 464–478.

Liu, G., & Xiong, Y. L. (2000). Oxidatively induced chemical changes and interactions of

mixed myosin, β-lactoglobulin and soy 7S globulin. Journal of the Science of Food and

Agriculture, 80(11), 1601–1607.

Loch, J. I., Bonarek, P., Tworzydlo, M., Polit, A., Hawro, B., Lach, A., et al. (2016).

Engineered beta-lactoglobulin produced in E. coli: Purification, biophysical and

structural characterisation. Molecular Biotechnology, 58(10), 605–618.

Losito, I., Stringano, E., Carulli, S., & Palmisano, F. (2010). Correlation between

lactosylation and denaturation of major whey proteins: An investigation by liquid

J.K. Keppler et al.

Food Hydrocolloids 110 (2021) 106132

chromatography-electrospray ionization mass spectrometry. Analytical and

Bioanalytical Chemistry, 396(6), 2293–2306.

Mackie, A. R., Husband, F. A., Holt, C., & Wilde, P. J. (1999). Adsorption of

β-Lactoglobulin variants A and B to the air–water interface. International Journal of

Food Science and Technology, 34(5-6), 509–516.

Mahn, A, Lienqueo, ME, & Asenjo, JA (2004). Effect of surface hydrophobicity

distribution on retention of ribonucleases in hydrophobic interaction

chromatography. Chromatogr A, 1043(1), 47–55. https://doi.org/10.1016/j.

chroma.2004.03.021.

Marti-Renom, M. A., Stuart, A. C., Fiser, A., Sanchez, R., Melo, F., & Sali, A. (2000).

Comparative protein structure modeling of genes and genomes. Annual Review of

Biophysics and Biomolecular Structure, 29, 291–325.

McLean, D. M., Graham, E. R. B., Ponzoni, R. W., & McKenzie, H. A. (1987). Effects of

milk protein genetic variants and composition on heat stability of milk. Journal of

Dairy Research, 54(2), 219–235.

Mercadante, D., Melton, L. D., Norris, G. E., Loo, T. S., Williams, M. A. K.,

Dobson, R. C. J., et al. (2012). Bovine b-lactoglobulin is dimeric under imitative

physiological conditions: Dissociation equilibrium and rate constants over the pH

range of 2.5–7.5. Biophysical Journal, 103, 303–312.

Mishyna, M., Chen, J., & Benjamin, O. (2020). Sensory attributes of edible insects and

insect-based foods – future outlooks for enhancing consumer appeal. Trends in Food

Science & Technology, 95, 141–148.

Norwood, E.-A., Chevallier, M., Le Floch-Fou�

er�

e, C., Schuck, P., Jeantet, R., &

Croguennec, T. (2016). Heat-induced aggregation properties of whey proteins as

affected by storage conditions of whey protein isolate powders. Food and Bioprocess

Technology, 9(6), 993–1001.

Panick, G., Malessa, R., & Winter, R. (1999). Differences between the pressure- and

temperature-induced denaturation and aggregation of beta-lactoglobulin A, B, and

AB monitored by FT-IR spectroscopy and small-angle X-ray scattering. Biochemistry,

38(20), 6512–6519.

Paul, A. A., Kumar, S., Kumar, V., & Sharma, R. (2019). Milk Analog: Plant based

alternatives to conventional milk, production, potential and health concerns. Critical

Reviews in Food Science and Nutrition, 1–19.

Pizzano, R., Manzo, C., Nicolai, M. A., & Addeo, F. (2012). Occurrence of major whey

proteins in the pH 4.6 insoluble protein fraction from UHT-treated milk. Journal of

Agricultural and Food Chemistry, 60(32), 8044–8050.

Ponniah, K., Loo, T. S., Edwards, P. J. B., Pascal, S. M., Jameson, G. B., & Norris, G. E.

(2010). The production of soluble and correctly folded recombinant bovine

β-lactoglobulin variants A and B in Escherichia coli for NMR studies. Protein

Expression and Purification, 70(2), 283–289.

Qin, Bin, Y., Bewley, Maria, C., Creamer, Lawrence, K., Baker, Edward, N., &

Jameson, Geoffrey, B. (1999). Functional Implications of Structural Differences

Between Variants A and B of Bovine Beta-Lactoglobulin. Protein Science, 8(1), 75–83.

Rade-Kukic, K., Schmitt, C., & Rawel, H. M. (2011). Formation of conjugates between

β-lactoglobulin and allyl isothiocyanate: Effect on protein heat aggregation, foaming

and emulsifying properties. Food Hydrocolloids, 25(4), 694–706.

Renard, D., Lefebvre, J., Griffin, M. C. A., & Griffin, W. G. (1998). Effects of pH and salt

environment on the association of [beta]-lactoglobulin revealed by intrinsic

fluorescence studies. International Journal of Biological Macromolecules, 22(1), 41–49.

Righetti, P., Gianazza, E., Gelfi, C., & Chairi, M. (1990). Gel electrophoresis of proteins: A

practical approach (2nd ed.). Oxford: Oxford University Press.

Roth, M. (1971). Fluorescence reaction for amino acids. Analytical Chemistry, 43(7),

880–882.

Ruprichov�

a, L., Kr�

alov�

a, M., Borkovcov�

a, I., Vorlov�

a, L., & Bed�

a�

nov�

a, I. (2014).

Determination of whey proteins in different types of milk. Acta Veterinaria Brno, 83

(1), 67–72.

Sagis, L. M. C., & Scholten, E. (2014). Complex interfaces in food: Structure and

mechanical properties. Trends in Food Science & Technology, 37(1), 59–71.

Scheidegger, D., Pecora, R. P., Radici, P. M., & Kivatinitz, S. C. (2010). Protein oxidative

changes in whole and skim milk after ultraviolet or fluorescent light exposure.

Journal of Dairy Science, 93(11), 5101–5109.

Schestkowa, H., Wollborn, T., Westphal, A., Wagemans, A., Fritsching, U., & Drusch, S.

(2019). Conformational state and charge determine the interfacial stabilization

process of beta-lactoglobulin at preoccupied interfaces. Journal of Colloid and

Interface Science, 536, 300–309.

Schultz, D. A., & Baldwin, R. L. (1992). Cis proline mutants of ribonuclease A. I. thermal

stability. Protein Science, 1(7), 831–958.

Semagoto, H. M., Liu, D., Koboyatau, K., Hu, J., Lu, N., Liu, X., et al. (2014). Effects of UV

induced photo-oxidation on the physicochemical properties of milk protein

concentrate. Food Research International, 62, 580–588.

Sethi, S., Tyagi, S. K., & Anurag, R. K. (2016). Plant-based milk alternatives an emerging

segment of functional beverages: A review. Journal of Food Science & Technology, 53

(9), 3408–3423.

Stafford, W. F., & Sherwood, P. J. (2004). Analysis of heterologous interacting systems by

sedimentation velocity: Curve fitting algorithms for estimation of sedimentation

coefficients, equilibrium and kinetic constants. Biophysical Chemistry, 108(1),

231–243.

Tagliavia, M., & Nicosia, A. (2019). Advanced strategies for food-grade protein

production: A new E. coli/lactic acid bacteria shuttle vector for improved cloning

and food-grade expression. Microorganisms, 7(5), 116.

Tamm, F., & Drusch, S. (2017). Impact of enzymatic hydrolysis on the interfacial

rheology of whey protein/pectin interfacial layers at the oil/water-interface. Food

Hydrocolloids, 63, 8–18.

Tenorio, A. T., Jong, E. W. M.d., Nikiforidis, C. V., Boom, R. M., & van der Goot, A. J.

(2017). Interfacial properties and emulsification performance of thylakoid

membrane fragments. Soft Matter, 13(3), 608–618.

To, BC, & Lenhoff, AM (2007). Hydrophobic interaction chromatography of proteins. I.

The effects of protein and adsorbent properties on retention and recovery.

J Chromatogr A., 1141(2), 191–205. https://doi.org/10.1016/j.chroma.2006.12.020.

Tolkach, A., & Kulozik, U. (2007). Reaction kinetic pathway of reversible and irreversible

thermal denaturation of beta-lactoglobulin. Le Lait, 87(4-5), 301–315.

Toro-Sierra, J., Tolkach, A., & Kulozik, U. (2013). Fractionation of

-lactalbumin and

β-lactoglobulin from whey protein isolate using selective thermal aggregation, an

optimized membrane separation procedure and resolubilization techniques at pilot

plant scale. Food and Bioprocess Technology, 6(4), 1032–1043.

Ui, N. (1971). Isoelectric points and conformation of proteins I. Effect of urea on the

behavior of some proteins in isoelectric focusing. Biochimica et biophysics acta, 229,

567–581.

US9924728B2, Patent, Pandya, R., Gandhi, P., Ji, S., Beauchamp, D., & Horn, L. (2018).

Food compositions comprising one or both of recombinant beta-lactoglobulin protein and

recombinant alpha-lactalbumin protein.

Uttinger, M. J., Heyn, T. R., Jandt, U., Wawra, S. E., Winzer, B., Keppler, J. K., et al.

(2020). Measurement of length distribution of beta-lactoglobulin fibrils by

multiwavelength analytical ultracentrifugation. European Biophysics Journal (in

press).

Uttinger, M. J., Wawra, S. E., Guckeisen, T., Walter, J., Bear, A., Thajudeen, T., et al.

(2019). A comprehensive Brownian dynamics approach for the determination of

non-ideality parameters from analytical ultracentrifugation. Langmuir, 35(35),

11491–11502.

Van Der Spoel, D., Lindahl, E., Hess, B., Groenhof, G., Mark, A. E., & Berendsen, H. J. C.

(2005). GROMACS: Fast, flexible, and free. Journal of Computational Chemistry, 26

(16), 1701–1718.

Verheul, M., Pedersen, J. S., Roefs, S. P. F. M., & Kruif, K. G. (1999). Association behavior

of native β-lactoglobulin. Biopolymers, 49(1), 11–20.

Vestergaard, M., Chan, S. H. J., & Jensen, P. R. (2016). Can microbes compete with cows

for sustainable protein production - a feasibility study on high quality protein.

Scientific Reports, 6(1), 36421.

van der Weele, C., Feindt, P., Jan van der Goot, A., van Mierlo, B., & van Boekel, M.

(2019). Meat alternatives: An integrative comparison. Trends in Food Science &

Technology, 88, 505–512.

Yagi, M., Sakurai, K., Kalidas, C., Batt, C. A., & Goto, Y. (2003). Reversible unfolding of

bovine beta-lactoglobulin mutants without a free thiol group. Journal of Biological

Chemistry, 278(47), 47009–47015.

Yan, Y., Seeman, D., Zheng, B., Kizilay, E., Xu, Y., & Dubin, P. L. (2013). pH-dependent

aggregation and disaggregation of native β-lactoglobulin in low salt. Langmuir, 29

(14), 4584–4593.

J.K. Keppler et al.