FORMATION OF CONJUGATED PROTEIN BY ELECTROSPINNING
CROSS-REFERENCE TO RELATED APPLICATIONS
[0 This application claims priority to U.S. provisional application Serial No. 61/619,996
filed April 4, 2012, hereby incorporated by reference in its entirety.
BACKGROUND
[02] Conjugated proteins, including protein-polysaccharide conjugates, play a significant role
in the structure and stability of many processed foods. One reaction that is particularly
important in the processed foods industry is the Maillard reaction. The Mai!lard reaction
is a nonenzymatic chemical interaction involving the condensation of an amino group and
a reducing group. The reaction results in the formation of intermediates that can later
polymerize to form brown, nitrogen-containing compounds known as mellanoidins.
[03] There are three major stages to the Maillard reaction. In the first stage, glycosylaniine is
formed then undergoes a rearrangement into an Amadori compound. n the second stage,
the amine group is lost and a carbonyl intermediate is formed. The carbonyl intermediate
undergoes dehydration or fission to form highly reactive carbonyl compounds. In the
final phase, the reactive carbonyl compounds react with other constituents of the food
product to form melanoidins.
[04] Products of the Maillard reaction are associated with positive attributes such as aroma,
taste, and color. However, the reaction can also lead to reduced nutritional value,
shortened shelf-life, and formation of undesirable compounds resulting in an off-taste
|05| Controlling the Maillard reaction is therefore critical in developing foods with improved
nutritional value. Previous ways of producing protein-conjugates have not been effective
for a variety of reasons. For example, dry incubation, which uses a lyophilization process
combined with heating, is slow and does not provide adequate yields. Consequently,
there is a need for more effective ways of controlling the production of conjugated
proteins.
SUMMARY
06] The features described herein generally relate to methods of using electrospinning to
produce conjugated proteins. Aspects of the features described herein relate to methods of
preparing dextran-conjugated whey proteins.
BRIEF DESCRIPTION OF THE DRAWINGS
Q7] The present disclosure is illustrated by way of example and not limited in the
accompanying figures in which like reference numerals indicate similar elements and in
which:
08] Figure 1 illustrates an example electrospinning apparatus on which various features of the
disclosure may be implemented.
09] Figure 2 illustrates beads on fibers with dextran (70 kDa) at 0.5 g/mL solvent.
10] Figure 3 illustrates fibers prepared from a solution of dextran (70 kDa) at 0.7 g/mL solvent.
11] Figure 4 illustrates smooth fibers prepared from a solution of dextran 70 kDa) at 0.8 g/mL
solvent.
1 ] Figure 5 illustrates an LM image of neat dextran electrospun fibers prepared from an
aqueous solution of dextran (40 kDa.) at .0 g/mL.
13] Figure 6 illustrates an LM image of neat dextran electrospun fibers prepared from the
aqueous solution of dextran ( 00 kDa) at 0.6 g mL.
14] Figure 7 illustrates the viscosity of dextran-whey protein isolate mixtures prepared with
different dextrans at the minimum electrospinnable concentration.
| 5| Figures 8A-F. Figures 8A-8C illustrates Scanning Electron Microscope (SEM) images of
dextran-whey protein isolate electrospun fibers prepared from various dextran sizes: 8A: 40
kDa, 8B: 70 kDa, and 8C: 100 kDa. Fiber diameters for the fibers in Figs 8A, 8B, and 8C
are shown in Figs. 8D, 8E, and 8F, respectively.
jl | Figure 9 illustrates stress-strain flow curves of aqueous dextran solutions at different
concentrations.
17| Figure 10 illustrates the effect of the mixing ratio on the flow behavior of dextran and whey
protein mixtures. The total solid content was 0.6 g/mL for each mixture.
[18| Figures 11A-H. Figures 11A-D illustrate SEM images of electrospun fibers prepared from
dextran solutions at different concentrations ( 11A: 0.3 g/mL solvent, 11B: 0.45 g/mL
solvent, C 0.6 g/mL solvent, and 11D: 0.7 g/mL solvent). Electrospinning conditions
were kept constant at: voltage = 20 kV, electrospinning distance = 18 cm and solution flow
rate = 12 L/ in. Fiber diameters for the fibers in Figs 11A-D are shown in Figs. 1E-H,
respectively.
19| Figures 2A- . Figures 12A-E illustrates SEM images of electrospun fibers prepared from
mixtures with different mixing ratios (by weight) of dextran (100 kDa) and whey protein
isolate (12A: 1:0, 12B: 0.8:0.2, 12C: 0.75 :0 .25; 12D: 0.67:0.33, and 12E: 0.5 :0 .5). The
concentration of all mixtures was 0.6 g/mL solvent and electrospinning conditions were
kept constant at: voltage = 20 kV, electrospinning distance = 18 cm and solution flow rate
= 12 m ,/h h. Fiber diameters for the fibers in Figs A-E are shown in Figs 12F-J,
respectively.
[20] Figure 13A shows 1R spectra of the mixture between dextran powder and whey protein
isolate powder at different mixing ratio. Figure 13 B shows the relationship between whey
protein isolate content and the ratio between peak absorbance at wavenumber of 1518 and
1004 cn .
[21 Figure 14 shows the appearance of electrospun films annealed at 60 °C and 74 %RH for
different times.
2 Figure 15 illustrates R spectra of: a) WPl-dextran in powder form; b) as-spun WPI-dextran
electrospun film; c) to f WPI-dextran electrospun films after annealed at 60 C and 74
%RH for 2, 6, 20 and 48 hours, respectively.
[23 Figure 16 illustrates the effect of annealing time on SDS-PAGE profiles of WPI-Dextran
conjugates in a presence of 2-mereaptoethanoI. Lane 1: WP solution; lanes 2-8:
electrospun WPI-dextran (40 kDa) film annealed at 60 °C 74%RH for 0, 2, 4, 6, 8, 16, 24
and 48 h, respectively.
[24] Figures 17A and 17B illustrate SDS-PAGE profiles of WPI-Dextran conjugates in a
presence of 2-mercaptoethanol. Fig. 7A is a protein stain. Fig 7B is a glycoprotein stain.
Lanes for both Fig. 17A and B are Lane 1: WPI solution; lanes 2-5: electrospun WPIdextran
(40kDa) film heated at 0, 8, 16, and 24 h, respectively; lanes 6-9: electrospun WPIdextran
( 00 kDa) film heated at 0, 8, 6, and 24 h, respectively.
[25] Figures ISA and 18B illustrate SDS-PAGE profiles of WTI-Dextran (70 kDa) conjugates:
Fig. A is a protein stain. Fig 18B is a glycoprotein stain. Weight ratio between dextran
and WPI in a electrospun films was 3:1. Lane 1: protein ladder; lanes 2-5: electrospun
films annealed at 60 °C, 74 %RH at 0, 8, 6 and 24 h, respectively.
[26| Figure 19 illustrates the Standard curve of TGX pre-cast ge used for the SDS PAGE gels.
Variables M W and d shown in the equation are molecular weight in kDa and traveling
distance in , respectively (R =99.86%). The center line and two outer lines show the
predicted values and 95% confidence-band, respectively.
[27| Figures 20A and 20B illustrate SDS-PAGE profiles of WPI-Dextran (100 kDa) conjugates
formed under different humidity and annealing times. Weight ratio between dextran and
WPI in all electrospun films was 3:1. Fig. 20A is a protein stain. Fig 20B is a glycoprotein
stain. Lane I : as-spun film; lanes 2-10: electrospun films annealed at 60 °C but with
different relative humidity: lanes 2-4 == 0 %RH; lanes 5-7 = 44 %R ; lanes 8-10 = 74
%RH. Annealing times were 8h: lanes 2, 5 and 8; 6h; lanes 3, 6 and 9 : 16h; and 24h lanes
4, 7 and 0 . Lane 0 is standard protein ladder
[28] Figures 2 1 and 2 B illustrate SDS-PAGE profiles of WPI-Dextran (100 kDa) conjugates
under constant humidity conditions. Eiectrospun films were prepared at different molar
ratios between dextran and WPI: lanes 1-4=4:2; lanes 5-8: 1:1; lanes 9-12: 2:1,
respectively. Fig. 21A is a . protein stain. Fig 21B is a glycoprotein stain. All eiectrospun
films were annealed at 60 °C and 74 %RH. Annealing times were: lanes 1, 5 and 9 : as-spun
films; lanes 2, 6 and 10: 8h: lanes 3, 7 and 11: 16h; lanes 4, 8 and 12: 24h. Lane 0 is
standard protein ladder. The dye used is specific to N-glyeosides and which are not
present in the protein or maltodextrin
[29] Figure 22A shows V absorbance spectra of eiectrospun samples prepared from mixtures
of WPI and dextran 40 kDa annealed at 60 °C and 74 %RH with different annealing times.
Figure 22B shows the relationship between absorbance peak at 282 nm of eiectrospun
samples and annealing time (WPI and dextran 40 kDa samples).
[30] Figure 23A shows UV absorbance spectra of eiectrospun samples prepared from mixtures
of WPI and dextran 70 kDa annealed at 60 °C and 74 %RH with different annealing times.
Figure 23B shows the relationship between absorbance peak at 282 nm of eiectrospun
samples and annealing time (WPI and dextran 70 kDa samples).
[31] Figures 24A and 24B shows the effect of solution concentration on UV absorbance of
eiectrospun samples prepared from mixtures of WPI and dextran 00 kDa Fig. 24A is 5
mg/n L and Fig. 24B is 3 mg/mL.
[32] Figures 25A and 25B. shows the absorbance peak at 282 nm of eiectrospun samples
prepared from mixtures of WPI and dextran 00 kDa at the molar ratio of 1:1 (FIG. 25A)
and 2:1 (FIG. 25B).
33 Figure 26 shows UV absorbance spectra of electrospun samples prepared from mixtures of
dextran 100 kDa and WPI with a molar ratio of 2 : .
3 Figure 27 shows the electrospun films prepared fro WPI and dextran 100 kDa with
different mixing ratios: A) 1:2; B) 1:1; and C) 2 : (molar ratio between dextran and WPI)
35| Figures 28 A-C illustrate lightness (filled circle) and yellowness (unfilled circle) of
electrospun films measured using a colorimeter at molar rations of :2 (A), :1 (B), and 2:2
(C).
36] Figures 29 A and B illustrate the NI spectra of electrospun films WPI-dextran 1OOkDa.
DETAILED DESCRIPTION
37] In an aspect of the disclosure, methods are provided to produce conjugated proteins using
an electro-spinning approach. In some aspects, the conjugate is a protein-carbohydrate
conjugate (i.e a protein covalently linked to one or more carbohydrates). In some aspects,
the conjugate is a protein-po!ysaccharide conjugate (i.e a protein covalently linked to one
or more polysaccharides). In particular aspects, the polysaccharide is a dextran and the
protein is a whey protein isolate (WPI).
a . Whey Proteins and Glycosylation
38] Whey proteins are key ingredients used in a wide variety of food products due to their
excellent functionality (e.g. foam and emulsion stabilization) and their high nutritional
content (e.g. high content of essential amino acids) [1]
39] Whey proteins are readily denatured during food processing due to intrinsically
encountered environmental conditions, such as high temperature, pressure and/or presence
of various acids. Improvement of whey protein stability has therefore been a key interest
for food manufacturers in order to improve their use in food applications. Glycosylation
between whey protein and dextran through the formation of Schiff base intermediate
products has been proved to be an effective method to improve the proteins' heat stabilities
[1] and emulsifying properties [3, 4]
Formation of protein-polysaccharide conjugates improved the freeze-thaw stability of the
emulsion, even after three cycles of freezing-thawing at - 8 C for 2 hours and +40 °C for
hours [5]
Non-chemical glycosylation of protein and polysaccharide can be conducted by dry heating
a mixture of protein powder and a powder containing reducing sugars at about 60 C and a
relative humidity of at least -44% for up to 4 days [6] . n dry heating, rather than applying
the heat directly to WP1 and dextran in their initial powder form, solutions of W and
dextran in liquid form are first prepared, followed by freeze drying to obtain a solid form of
a mixture, which is more suitable for the subsequent glycosylation reaction.
An alternative method of glycosylating WP is to heat dextran and whey protein isolate
solutions at 60 C and p -6.5 for 48 hours, so called wet-heating. See, for example U.S.
Published Application 2009031 1407. Wet heating has a substantially shorter reaction time
than dry-heating, and the advanced Maillaixl reaction products, which yield dark pigments
are not as readily formed as in the dry heating method. Neither method has met with
widespread use because of the expense involved and because both methods had low yields
(<5%).
ELECTROSPINNING
Electrospinning is a form of electro-depositioning and is a versatile technique that can
produce fibers from synthetic and/or natural polymers by using a high voltage electric field
(e.g. 15 to 25 kV). Electrospinning may be with a needle or needleless. A typical
electrospinning set up consists of a . solution container equipped with a spinnerette, a
collector plate used to collect the deposited fibers and a high voltage unit to generate
electric fields between the spinneret and the collector plate. See Fig. 1. A solution
container may be a syringe equipped with a conductive needle, which can be used both as a
spinneret and an electrode.
] The flow rate of the polymer solution can be controlled using a syringe pump. Once the
polymer solution is electrically charged via an electrode, the shape of a polymer solution
droplet at the spinneret tip will transform to a Taylor cone shape. f the voltage is high
enough to overcome surface tension forces, thin polymer jets will be ejected from the
Taylor's cone tip. The ejected polymer jets dry rapidly due to the small dimension of the
jet, and dry solid fibers can be collected from a collector plate. The average diameters of
eiectrospun fibers are typically around a few hundred nanometers.
] Polymer chain entanglements are required to successfully electrospin a target solution into
fibers. Polymer chain entanglements prevent polymer jets from breaking up due to electric
stretching forces during electrospinning. If the polymer concentration is too low, polymer
chain overlapping in the solution is absent. When the concentration increases to a critical
concentration, c*, chain entanglement is initiated. The degree of chain entanglement
depends on both polymer concentration and molecular weight, (h ) 0] , and can be
determined by Eq. . [12]:
¾
] where c is the polymer concentration in the solution, Mw is the molecular weight of the
polymer in the solution, and M is the polymer entanglement molecular weight or the
average molecular weight between entanglement junctions.
] Eq. 2.1 establishes that the higher the polymer molecular weight, the lower the polymer
concentration required in order to maintain a constant entanglement number, i.e., maintain
electrospinnability of the solution.
PROTEINS AND CARBGHYDRATES/POLYSACCHARIDES
] Whey protein is a key ingredient in food products. Whey proteins offer good foam and
emulsion stability and also have a high content of essential amino acids. The preparation of
whey proteins is known in the art. Typically, the milk cream is centrifuged to remove the
cream or fat, then the caseins are precipitated allowing the whey proteins to be isolated by
additional centrifugation, filtration and/or ion exchange steps. A typical whey protein
isolate (WPI) may contain b-lactoglobuiin (-55%), a-iactalbumin (-25%),
immunoglobulin (-15%), bovine serum albumin (~4%) and lactoferrin (-2%).
While WPI is a preferred glycosylation target, a variety of proteins may be selected for
electrospinning. In some aspects, only one type of protein is used. In other aspects, the
protein may be a mixture of one or more proteins.
Any soluble animal or plant protein that ca be electrospun and conjugated (reacted with
saccharides) may be used. Suitable proteins include plant-based proteins, animal-based
proteins, and microbial-based proteins. For example, suitable plant-based proteins may
include Soy (Concentrate/Isolate), Pea (Concentrate/isolate), Wheat (Gliadin, Gluten),
Lentil, Beans, Cora (Zein), Oat, Barley, Amaranth, Rice, Buckwheat, Farro, Flaxseed,
Quinoa, Rye, Sorghum, Teff, Sunflower, and Nuts (Peanuts, almonds, pecans). Suitable
animal-based proteins may include Dairy (Whey and casein), Fish protein, Animal
protein (Beef, pork, veal, poultry. . .), and Egg protein. Suitable microbial-based proteins
may include alga, funagal, and bacterial.
Carbohydrates having a reducing functional group may be utilized with the instant process.
n particular, a suitable carbohydrate either has an aldehyde group or has the capability of
forming an aldehyde in solution through isomerism. Suitable carbohydraes include agar,
agarose, alginate, alginic acid, alguronic acid, alpha glucan, amylopectin, amylose,
arabinogalactan, arabinoxylan, beta-glucan, BioCell collagen, callose, capsulan,
carrageenan, cellodextrin, cellulin, cellulose, chitin, Chitin nanofibril, chitosan,
chrysolaminarin, curdlan, cyclodextrin, DEAE-sepharose, clextran, dextrin,
exopolysaccharide, alpha-cyciodextrin, Fibersol, ficoll, fructan, fucoidan,
gaiactoglueomannan, gaiaetomannan, gellan gum, glucan, glucomannan, glyeocalyx,
glycogen, hydrolyzed guar, guar gum, gum arable, hemicellulose, homopolysaccharide,
hypromellose, icodextrin, inulin, inulose, kefiran, konjac, laminarin, lentinan, levan
lichenin, locust bean gum, MatrixDB, mixed-linkage, mean, modified starch,mucilage,
natural gum, oxidized cellulose, paramyion, peetic acid, pectin, pentastarch, pleuran,
polydextrose, polygiycol alginate, polysaccharide, polysaccharide peptide, rof ΐ n h,
pulluian, schizophyllan, sepharose, sinistrin, sizofiran, soy fiber, sugammadex,
unhydrolysable glucose polymers, welan gum, xanthan gum, xylan, xvloglucan, yucca or
yucca/quiilaia. extracts, and zymosan.
A particular category of suitable carbohydrates is polysaccharides.
Saccharides such as polysaccharides are polymers of saccharides. Oligosaccharides are
polymers of 2-200 saccharides. Polysaccharides are longer polymers than
oligosaccharides. Suitable polysaccharides may be polymers of 201 to 2500 saccharides.
Suitable polysaccharides include dextrans. A dextran means a complex, branched
polysaccharide comprising multiple glucose molecules joined into chains of varying
lengths (e.g, from 0 to 50 kDa). The straight chain consists of ®6 glycosidic linkages
between glucose molecules. The branched chain may contain —»4 linkages, ®2
linkages and/or a l®3 linkages.
Saccharides (sugar types) suitable for conjugation conformation include reducing
monosaccharides such as glucose, fructose, glyceraldehyde and galactose. Many
^saccharides, like lactose and maltose, also have a reducing form, as one of the two units
may have an open-chain for with an aldehyde group. n glucose polymers such as starch
and starch-derivatives like glucose syrup, maltodextrin and dextrin the macromolecule
begins with a reducing sugar, a free aldehyde. More hydrolyzed starch contains more
reducing sugars. The percentage of reducing sugars present in these starch derivatives is
called dextrose equivalent (DE).
n some embodiments, the carbohydrates, such as dextrans, have a molecular weight range
between about 10 kDa and about 500 kDa. n other embodiments, the dextrans have a
molecular weight range between about 40 kDa and about 00 kDa, or between about 70
kDa and about 100 kDa.
|57| "About," as used herein, means +/- 10% of the numerical value.
[58] Any suitable electrospinning methods and devices may be used so long as the following are
provided: a high power voltage supply, a reservoir for the polymer solution, electrodes (one
grounded), and a receiver plate. Suitable electrospinning devices include, but are not
limited to, Needle-less and Near Field.
[59] Electrospinning provides substantial advantages over the previous methods of conjugating
proteins. The present electrospinning method facilitated glycosylation in much shorter
annealing times, and with a greater yield.
|60| In eiectrospun WPl-dextran fibers, for example, the physical state of the two polymers is
such that a higher molecule-to-molecule larger contact number is achieved for at least three
reasons. First, the polymers are more uniformly distributed since electrospinning causes a
molecular alignment due to stretching and bending motions. Second, a phase separation is
prevented because of the rapid evaporation of solvent. Finally, the small diameters of
fibers results in a densely packed fiber composed of WP and dextran molecules.
|6ί | Thus, without being bound by theor}' it is thought that the electrospinning process causes
molecules to be well aligned and that stay intact due to polymer chain entanglements,
resulting in particularly good yield and reduced cost.
[62] A variety of parameters may be varied to optimize fiber formation during electrospinning.
Parameters that affect fiber formation include the dextran concentration, mixture viscosity,
protein-to-polysaccharide ratio in the mixture, time allowed for annealing.
3 The following description focuses on one aspect of the invention, namely dextran-whey
protein. t is to be understood that the process can be extended to any suitable protein and
carbohydrate.
i . Dextran Concentration and Fiber Formation
|64| Dextran was responsible for the electrospinnability of dextran-whey protein mixtures while
simultaneously serving as the reducing sugar reagent for the conjugate formation.
A wide range of dextran molecular weights from 10-440 kDa have been used to form
conjugates with proteins. Lower molecular weights typically give higher levels of
conjugates. For electrospinning, high concentrations are required when low molecular
weight polymers are used. Unfortunately, high concentrations result in highly viscous
solutions, which are not capable of electrospinning.
[66] The ability of dextran to form fibers is related to dextran concentration and size. All
dextran (Mw = 100 kDa) solutions with the concentration down to 0.45 g/mL exhibited
shear thinning flow behaviors. For 100 kDa dextran, smooth and continuous fibers
required a concentration of 0.1 g/mL or higher or 0.6 g/mL or higher. Thus, in some
aspects for 100 kDa dextran, the mixture for electrospinning contains at least 0.6 g/mL, at
least 0.8 g/mL, or at least 1.0 g/mL dextran. An upper limit for dextran may be determined
by the viscosity of the mixture and depends on the type of polymers such as 5 g/mL.
|67| The protein and polysaccharide may be combined in a buffer for electrospinning. For
example, the mixture for electrospinning may be prepared in phosphate buffer. The pH of
the buffer is between 6.0 and 7.0: typically, the buffer pH is 6.50±0.07
ii. Viscosity
|64| Viscosity is an important parameter for efficient electrospinning. When viscosity is too
high, electrospinning efficiency is negatively impacted. For example, viscosity for a
solution prepared from 40kDa dextran was about 1.8 Pa.s (Poises). This level of viscosity
was a disadvantage for several reasons. First, solution preparation was difficult because the
solution became "sticky " Second, a thick layer of foam formed during solution
preparation due to high content of protein in the mixture (0.25 g/mL solvent). The solvent
may be any suitable solvent in which the polymer may be dissolved. Ideally the solvent
should rapidly evaporate and should not be flammable. A suitable solvent is water. A long
relaxation time was therefore required to remove the foam layer from the mixture.
Moreover, clogging took rapidly place during the eiectrospinning, resulted in the lowest
observed productivity.
[6 Viscosity may be measured by any suitable method using any suitable viscometers and
rheometers such as, but not limited to, a Brookfield Viscometer. Generally the viscosity
should be along a desired flow curve and is not limited to any single value.
[66] n contrast, a solution prepared from 00 kDa dextran-—the largest dextran tested— had the
lowest viscosity. The lower viscosity eased solution preparation, decreased foam layer
formation, reduced clogging frequency.
[67 Suitable viscosities may be obtained by using dextran sizes above 40 kDa. For example,
such mixtures may contain dextrans between 50 kDa and about 00 kDa. n aspects,
mixtures of dextran sizes between 50 kDa and about 00 kDa may be used to obtain the
desired viscosity levels. In particular aspects, the dextran has a molecular weight about 00
kDa.
iii. Fiber Size and Formation
[68] It is believed that conjugation of polysaccharide to the protein is enhanced when bead
formation is reduced or eliminated. Beads are considered a defect and hence undesirable.
Fibers with little or no beads are preferred. In aspects of the disclosure, the fibers contain
no beads as determined by Scanning Electron Microscopy.
[69] Fiber diameter may affect conjugation. Smaller diameters of fibers results in a densely
packed fiber composed of WP and dextran molecules. Conjugation is promoted by dense
packing of the WP and dextran molecules. n some aspects, fiber diameter in some
aspects is about 00 ran and about 500 nm. In other aspects the fiber diameter is about
100 nm and about 300 nm. In yet other aspects, fiber diameter is between about 150 nm
and about 250 nm. Fiber diameter may be measured as described in Example 1.
iv. Molar Ratio of Protein and Dextran
Protein content of the fiber also affects both fiber formation and conjugation. When the
protein content of the mixture increases, fiber diameter decreases. See, for example.
Example 7. When the protein content was raised to 33% wt or higher, beads were formed.
Thus, to reduce or eliminate beading on fibers, protein content in the mixing solution may
be up to about 25% wt. To obtain these percentages, the molar ratio (weight/weight) of
polysaccharide to protein in the aqueous solution used for electrospinning may be as in
Table 1.
Table . Correspondence between dextran, protein, and bead formation
Additional details regarding protein content in films is given in Table 9 .
Formation of glycoprotein, measured by gel electrophoresis is enhanced when the ratio of
dextran: WPI increases. For example,
Thus, to achieve good fiber formation and conjugation the molar ratio of polysaccharide to
protein is above 2 . In some aspects, the ratio is about 3:1, about 4:1, about 5:1, about 5:1
about 5:1, about 5:1, about 5:1, about 5:1, about 5:1, about 5:1. Accordingly in particular
aspects the range of ratios may be about 3:1 to about 10:1 . In other aspects, the ratio may
be about 3:1 to about 4 : .
iv. Annealing Conditions
Electrospinning brings molecules close together. 'Then annealing provides a temperature
induced reaction between the molecules. Annealing, as used herein, is the conjugation
process whereby covalent bonds are formed between the protein and the polysaccharide.
Annealing may be enhanced by using a prolonged incubation period. For example, the
annealing time may have a lower limit 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12
hours, 14 hours, 16 hours, 8 hours, or 24 hours. Annealing time may have an upper limit
of 4 hours, 6 hours, 8 hours, 0 hours, 12 hours, 14 hours, 6 hours, 8 hours, 24 hours, or
28 hours. Often annealing time is between 2 hours and 4 hours. The color of films became
slightly darker when longer annealing times were used especially when films were
annealed for more than 24 hours.
v. Effect of dextran size on Conjugation
] Glycosylation was slower when dextran with 100 kDa was used to prepare electrospun
film. However, the size of glycoproteins formed between WP and dextran 100 kDa was
larger than -280 kDa while that formed from WPl and dextran with 40 and 70 kDa were
larger than ~70 and -200 kDa, respectively.
vi. Effect of humidity on glycosylation
| The methods may be performed at particular humidity levels to enhance glycosylation.
Higher humidity may enhance glycosylation, as determined by the more intense of
glycoproteins band observed in glycoprotein-stained gel . There was much less intensity of
glycosylation bands in glycoprotein-stained gel when samples were annealed under low
humidity. Enhanced glycosylation was obtained at 74% R i (Fig. 20A and B; Lanes 8-10)
compared to 0 %RH (Fig. 20A and B; Lanes 2-4) and 44 %RH (Fig. 20A and B; Lanes 5-
7). Thus, in some aspects, the relative humidity is at least 44%. In other aspects, the
relative humidity is between 45% and 75% or 65% and 75%: in yet other aspects, the
relative humidity is 70% to 80%.
] The temperature is typically about room temperature; however, any suitable temperature
between 0 C and 70 °C is contemplated
a . Assessing Conjugation
] The appearance of WPI-Dextran conjugates may be monitored by any suitable approach.
For example, gel electrophoresis may be a preferred way to verify the presence of
glycoprotein in annealed electrospun films. FT R did not clearly show a difference
between R spectra from as-spun films and annealed films and may therefore be less useful .
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The disclosures of patents, published applications, and journal articles cited above are
hereby incorporated by reference
Notwithstanding the claims, the invention is also defined by way of the following
clauses:
1. A method of preparing a carbohydrate-protein fiber or particle via electro-spinning
comprising steps of
preparing an aqueous solution comprising a carbohydrate and a protein,
applying a voltage of 15 to 25 kV to the solution,
collecting the fiber on a collecting plate.
2. The method of clause 1 wherein the electro-spinning is needleless.
3. The method of clause 1 or clause 2 wherein the carbohydrate has an aldehyde group
or forms an aldehyde group through isomerism.
4. The method of clause 1 or clause 2 wherein the carbohydrate is a dextran.
5. The method of claim 4 wherein the dextran molecular weight is between about 0
kDa and about 500 kDa.
6. The method of claim 4 wherein the dextran is present at concentration of 0.1 g/mL
to about 5.0 g/mL.
7. The method according to any of the preceding clauses wherein the protein is
selected from microbial, animal, dairy, and vegetable.
8. The method according to any of the preceding clauses wherein the protein is a whey
protein isolate (WPI).
9. The method according to any of the preceding clauses wherein the aqueous solution
comprises carbohydrate and protein at a molar ratio (w/w) from 50: 1 to 1:50.
0. The method according to any of the preceding clauses wherein the aqueous solution
comprises carbohydrate and protein at a molar ratio (w/w) from 3:1 to 1:10.
11. The method according to any of the preceding clauses wherein the aqueous solution
comprises dextran and WP .
12. The method according to any of the preceding clauses further comprising the step
of incubating the fiber at a relative humidity of at least 45%, for up to 24 hours, whereby a
conjugated film is formed.
13. The method of clause 2 wherein the relative humidity is between 65% and 75%.
4. The method of clause 2 wherein the temperature is in the range of 10-70 °C.
15. The method according to any of the preceding clauses wherein the fiber diameter is
about 100 ntn to about 500 nm.
16. The method of claim 15 wherein the fiber diameter is about 150 n to about 250
nm.
17. A method of preparing a polysaccharide -protein fiber by electro-spinning
comprising steps of
preparing an aqueous solution comprising 100 kDa dextran and a whey protein
isolate, wherein the dextran and the whey protein isolate are present in a molar ratio (w/w)
between 3:1 and 10:1,
applying a voltage of 15 to 25 kV to the solution whereby a fiber is created,
collecting the fiber on a collecting plate, and
incubating the fiber at a relative humidity of at least 45% for between 4 and 24
hours, whereby a conjugated film is formed.
18. The method of claim 17 wherein the relative humidity is between 65% and 75%.
19. The method of clause 7 or clause 8 wherein the incubation is between 4 and 8
hours.
20. The method according to any of clauses 16 - 8 wherein the temperature is in the
range of 10-70 °C.
21. The method according to any of clauses 16 -18 wherein the electro-spinning is
needleless.
EXAMPLE 1
Materials and Methods
a . Dextrans
De tra s with average molecular weight of about 40, 70 and 100 kDa, produced y
Leuconostoc spp., were purchased from Sigma-Aldrich (Steinheim, Germany). B P O©
whey protein isolate was supplied from Davisco Food Internationa], Inc. A materials were
used without further purification.
Dextran powder and/or whey protein isolate at various mixing ratios (1:0, 0 8:0.2, 0.75:25,
0 67:0.33 and 0.5:0.5 by weight) were dissolved in 30 M phosphate buffer solution. The
solution was stirred on a magnetic stirrer overnight at room temperature (-22 oC) to ensure
a complete dissolution of the mixture. The solution viscosity, conducti vity, density and pH
were measured using a rheometer (Physica, MCS 300, Ashland, VA), a microprocessor
conductivity meter (WTW, LF537, Weiheim, Germany), a density meter (Anton Paar,
DMA 35 , Graz, Austria) and a pH meter (WTW, inolab, Weiheim, Germany),
respectively. Final pH of all solutions was 6.50±0.07, which was previously reported as a
suitable pH for whey protein isolate-dextran conjugates formation [7j.
b. Electrospinning
The electrospinning set up is shown in Figure 1. The target solution was loaded into a . 10-
mL glass syringe equipped with a . stainless steel needle (inner diameter of 0.9 mm). The
syringe was then loaded into a syringe mounted on a syringe pump (Harvard Apparatus
I IP, Holliston, MA). The needle tip was connected to a high voltage D.C. power supply
(Gamma High Voltage, ES30P-5W, Ormond Beach, FL). The grounded electrode was
connected to a stainless steel collector plate. The distance between the needle tip and the
collector plate was maintained at 18 cm. The solution flow rate was kept constant at 12
jjL/min. The solution was electrically charged at about +20 kV. The collected electrospun
films were frozen at -28 C before further investigations.
Typically the voltage during electrospimiing is 5 to 20 kV and not above 25 kV If the
voltage is too high, there will be arcing (sparks). The amperage is increased until fiber
formation is achieved.
c . Morphology Analysis
The electrospun fibers were investigated for their morphology using a ZEISS ORIGA
cross-beam field emission scanning electron microscope with focused ion-beam
(Oberkochen, Germany). The films were vacuum-dried and gold sputter-coated for about 4
minutes. The average diameters of the electrospun fibers were determined by image
analysis using Image J (National Institutes of Health, Bethesda, MD).
d. Film characterization by FTIR
The electrospun films were investigated through the use of a Fourier transform infrared
spectrometer equipped with a universal attenuated total reflectance (ATR-FT R) accessory
(Perkin Elmer, Spectrum 00, Beaconsfield, UK). The infrared spectra were collected
within a wavelength range of 4000-650 cm 1, with a 4 cm 1 resolution and 10 scans, in
transmission mode.
e . SDS-PAGE gels
TGX pre-cast gels (Tris-HCl Gel, 4-20% linear gradient, 15 wells) and protein stainingdestaining
solutions (Bio-Safe Coomassie Stain and Coomassie Destain) were purchased
from Bio-Rad Laboratories. Standard protein ladder (PageRulerTM Plus Prestained Protein
Ladder 26619, 10-250 kDa) and glycoprotein staining kit (Pierce® Glycoprotein Staining
Kit) were purchased from Thermo Scientific, Pierce Biotechnology (Germany). The
standard protein ladder was kept at -28 °C before used. Potassium carbonate ( C0 3) and
sodium chloride (NaCl) were purchased from Sigma-Aklrich (Steinheim, Germany).
f . Heat Treatment
[89] About 5-10 n g of eleetrospun films were placed in a controlled humidity chamber. The
chamber was pre-equilibrated in a hot-air oven (Memmert, Model 400, Schwabach,
Germany) to allow the control volume to reach equilibrium temperature at 60 °C. The
relative humidity inside the control volume was about 0 %RH (using silica gel), 44 %RH
(using saturated KC0 3 solution) or 74 %RH (using saturated NaCl solution).
[90] Annealing provides conjugation of the protein and saccharide followed by a Malliard
Reaction.
g. Analysis of WPI-Dextran conjugate interaction studied by SDS-PAGE
[91] Sample buffer was prepared by dissolving all chemical compounds listed in Table 2 in
deionized distilled water to the final volume of 100 mL and frozen at -20 C before use.
[92] Table 2 Composition of sample buffer (200mL)
Electrode buffer was prepared by dissolving the compounds listed in Table 3 in deionized
distilled water to a final volume of 5000 mL. The buffer was stored at 4 C before use.
Table 3 Composition of electrode buffer ( 000 mL)
SDS 10% 0 mL
Glycine 14.4 g
] Samples were prepared for gel electrophoresis using a modified approach from the method
previously described by Zhu et a . (2008). First, heat-treated electrospun films were
dissolved in deionized distiiled water at a concentration of 80 g (of protein content)/! 5 L.
The aqueous solution was centrifuged at 16000 g for 15 min at 22 °C. The supernatant was
collected and further diluted with sample buffer to a final protein concentration of 40 ug
/Ί 5m . The diluted solution was then heated at 95 °C in water bath for at least 5 min to
break disulfide bonds and dissociate proteins (reducing conditions). The solution was then
kept cooled.
h. Gel electrophoresis
] SDS-PAGE was performed on a Min -PROTEAN Tetra Cell according to Laemmli (1970).
Reducing SDS-PAGE analyses were carried out on TGX pre-east gels (Tris-HCl Gel, 4-
20% linear gradient, 15 wells, Bio-Rad Laboratories). 10 mIAn of sample solution was
loaded into each well. Electrophoresis was run for about 40 min at a constant voltage of
200 V at room temperature. Two gels were run at the same time. After electrophoresis, one
gel was stained for protein and another was stained for glycoprotein to detect the presence
of WPI-dextran conjugates.
] For protein staining, gels were stained for protein and destained with Bio-Safe Coomassie
Stain and Coomassie Destain (Bio-Rad, Laboratories), respectively. The staining process
was carried out according to the manufacture's instruction. Briefly, the gels were washed
with an excess amount of deionized distilled water and transferred to the staining container
where the Bio-Safe Coomassie Stain was added to completely cover gels for at least i hour.
Then gels were destained in Coomassie Destain over night at room temperature.
] For glycoprotein staining, Pierce® Glycoprotein Staining Kit (Thermo Scientific, Pierce
Biotechnology) was used. Gels were fixed in 100 mL of 50% methanol for 30 minutes and
washed by gently agitating with 00 mL of 3% acetic acid for 10 minutes two times. Then
gels were immersed in oxidizing Solution for 5 minutes, washed in 00 mL 3% acetic
acid for 5 minutes (3 times), stained with Glycoprotein Staining Reagent for 15 minutes
and transferred to the reducing solution for 5 minutes, under gentle agitation. Finally, gels
were washed with an excess amount of 3% acetic acid followed by washing with deionized
distilled water.
i . Study of color development during annealing using UV-visible spectroscopy
Annealed electrospun films were dissolved in deionized distilled water to a concentration
of 5 mg/mL. The solution was shaken vigorously using a vortex for about two minutes and
kept still for about twenty minutes and then it was shaken again using a vortex to ensure a
complete dissolution. Ail solutions were used immediately after preparation.
j . UV-spectroscopy
V measurements were carried out on a UV-VIS-NIR Spectrometer with 60 m internal
sphere (PerkinElmer, Lambda 750S, Germany). 1 ml, of sample solution was loaded into
a semi-micro cuvette (Brand, PLASTIBRAND®, Germany). The sample was used for a
wavelength scan from 250 to 350 nm and the presence of conjugates was observed at
around 280 nm.
k. NIR spectra of as-spun and annealed electrospun films
NIR spectra of ultrafine-fibrous mats prior to and after heat treatment were investigated
using a UV-VIS-NIR Spectrometer with 60 mm internal sphere (PerkinElmer, Lambda
750S, Germany). The spectra were collected over a wavelength range of 250-2500 cm 1 in
reflectance mode, with a 4 cm resolution.
EXAMPLE 2
Preparing Electropun Fibers from Dextran
Dextrans with average molecular weight of about 40, 70 and 00 kDa were electrospun and
the morphology of the resulting fibers were investigated as described in Example 1
i . 70 kDa Dextran
[104] Dextran (70 kDa) gave a variety of morphologies depending on the concentration. At 0.5
g/mL (-33% w/w) beads were mainly produced with some short fibers (Figure 2).
Increasing the concentration to 0.7 g/mL (-41% w/w) resulted in fewer beads and
continuous fibers (Figure 3). Further increase in the dextran concentration to 0.8 g/mL
(-44% w/w) led to smooth fibers (Figure 4).
ii. 40 kDa Dextran
[105] For 40 kDa dextran the minimum electrospinnable concentration increased to 1 g/mL
solvent ( 50%w ). The fiber morphology was smooth and continuous (Figure 5). The
eiectrospinning conditions were kept constant at: voltage = 20 kV, electrospinning distance
= 18 cm and solution flow rate = 12 m / h.
iii. 100 kDa Dextran
106| For !OOkDa Dextran, the minimum dextran concentration decreased to 0.6 g/mL (-38
%w/w) in order to produce smooth iibers under the same electrospinning condition (Figure
6).
[107] Based on these data the minimum electrospinning concentration of dextran solutions was
calculated as in Table 4.
[ 08] Table 4 . Minimum Electrospinning Concentration of Dextran Solutions.
[1091 EXAMPLE 3
Viscosity Analysis of WPI-Dextran Mixtures
Generally, a polymer solution can be electrospun only within an appropriate range of
viscosities (1-20 poises) [14]. f the viscosity is too high, clogging may take place during
eiectrospinning [15]. Viscosity depends on polymer concentration and polymer molecular
weight. Mixtures of dextran-whey protein isolate were prepared and viscosities of the
mixtures were examined. The concentration of the mixture depended on dextran molecular
weight. The total concentration was the same as the minimum electrospinnable
concentration of dextran solution shown in Table 4.
The mixing ratio between dextran and whey protein isolate was kept constant at 3:1 (by
weight). The mixture formulations are summarized in Table 5.
Table 5. Dextran and whey protein isolate mixture formulation
Figure 7 shows the viscosity of these mixtures. The viscosity of mixture number 1 was
approximately 1.8 Poise (Pa.s) and about 2 - 5 times higher than the viscosity of the
mixtures number and 3, respectively.
The high viscosity of the solution prepared from 40 kDa dextran caused two problems.
First, the solution was difficult to prepare and to handle since the solution was not only
viscous but also very sticky. A thick layer of foam (-60% of total height) formed during
dissolution of the mixture, likely due to very high content of whey protein in the solution.
After overnight stirring, it was necessary to leave the solution in a vacuum chamber in
order to remove the air from the protein-air interface. Second, clogging rapidly took place
during eiectrospinning due to the high viscosity of the solution.
As a result, the productivity was the lowest in case of the mixture prepared from the lowest
molecular weight dextran. Accordingly, it was more convenient to prepare and to
eiectrospin the mixture prepared from 100 kDa dextran, which had the lowest viscosity
(about 0.4 Pa.s).
5| EXAMPLE 4
Morphology Analysis of Electrospun Fibers
6] Dextran-whey protein isolate electrospun fibers were prepared as described in Table 1 to
which was added whey protein isolate into dextran solution at a mixing ratio of 3:1
(dextran-whey protein isolate). Figure 8 shows selected SEM images of dextran-whey
protein isolate electrospun fibers. For all samples, fibers were smooth and no beads were
observed. See Figs. 8A-C. These data showed that the polymer concentration of each
solution had been appropriately selected, and that the incorporation of whey protein isolate
into dextran solution at a mixing ratio of 3:1 (dextran-whey protein isolate) did not
negatively affect the eiectrospinnability of the mixture.
7| The range of fiber diameters corresponding to Figs. 8A-C is shown in Figs 8A-F. The
diameters of the dextran-whey protein isolate fibers prepared from 40, 70 and 100 kDa
dextran were 4 1 80, 247±82, 173±72 nm, respectively. The difference in fiber
diameters may be due to the difference in solution viscosities.
8| The viscosity of the mixture prepared from 40 kDa dextran was highest because of the
large minimum electrospinnable concentration. The mixture prepared from 40 kDa dextran
displayed rapid clogging was observed and a very large average fiber diameter. This could
be due to the high viscosity n contrast, the mixture of 100 kDa dextran/whey protein
isolate mixture was easier to prepare and to handle, had a lower clogging frequency and
yielded smaller fiber diameters.
9| Accordingly, dextrans between 40 kDa and 100 kDa are useful for forming fibers, with
dextrans about 100 kDa being preferred.
1201 EXAMPLE 5
Properties of Mixtures Containing Dextran and VVP
] Dextran powder and/or whey protein isolate at various mixing ratios (1:0, 0.8:0.2, 0.75:25,
0.67:0.33 and 0.5:0.5 by weight) were dissolved in 30 mM phosphate buffer solution. The
solution was stirred on a magnetic stirrer overnight at room temperature ( 22 °C) to ensure
a complete dissolution of the mixture. The solution viscosity, conductivity, density' and pH
were measured using a rheometer (Physica, MCS 300, Ashland, VA), a microprocessor
conductivity meter (WTW, LF537, Weiheim, Germany), a density meter (Anton Paar,
DMA 35N, Graz, Austria) and a pH meter (WTW, Inolab, Weiheim, Germany),
respectively. Final pH of all solutions was 6.50±0.07
] The effects of dextran concentration and mixing ratio between dextran and whey protein
isolate on solution properties and fiber morpho logics were investigated.
] Figure 9 shows the flow behavior of aqueous dextran solution. The viscosities of the
solutions having concentrations of 0.7, 0.6, 0.45 and 0.3 g/niL solvent were approximately
1.4, 0.7, 0.3 and 0.1 Pa.s, respectively. Table 6 shows the consistency coefficient and the
flow behavior indices of all solutions. The first three solutions (0.7, 0.6 and 0.45 g/mL
solvent) exhibited shear thinning behavior as confirmed by the flow behavior index, n <
At the concentration of 0.3 g/mL solvent, the flow behavior of the solution became
increasingly Newtonian, which indicated a significant decrease in polymer chain
entanglement.
] Table 6 Consistency coefficient and the flow behavior indices.
] k and n values are Averages +/- Standard Deviation of duplicate measurements.
When WPI was added into the dextran solution, mixture viscosity decreased significantly.
Figure 10 shows the effect of WPI content on the viscosity of the dextran-whey protein
isolate mixture, compares with pure dextran solution. Higher WPI content correlated with
lower mixture viscosity. This might be because the globular structure of whey protein
isolate decreased the degree of chain entanglement of dextran in the mixture: and resulted
in a decrease in mixture viscosity. The flow behavior indices of the mixtures decreased
with an increase in protein content (Table 7. At the mixing ratio of 0.5:0.5, the mixture
seemed to have Newtonian flow behavior, similar to the dextran solution that had the
lowest concentration of 0.3 g/niL solvent.
Table 7 Consistency coefficient (k) and flow behavior index (n) of dextran solution and
dextran (100kDa)-whey protein isolate mixture
k and n values are Averages +/- Standard Deviation of duplicate measurements.
In contrast to the viscosity, the mixture's electrical conductivity increased with higher
protein content (Table 8). The WPI has a net negative charge because mixture pH was
about 6.5 and the isoelectric point (p ) of whey protein is 5.2. The polar structure of whey
protein isolate thus increased the total mobility of ions in the mixture, resulting in the
higher electrical conductivity.
Table 8 Effect of whey protein isolate content on mixture's electrical conductivity
4 0 67 0.33 1.26±0.02
5 0.50 0.50 1.51±0.03
*The values reported are the average value ± standard deviation of duplicate measurement.
EXAMPLE 6
Morphology of Fibers Electrospun from Dextran and Dextran/WPI Mixtures
a . Dextran Only Fibers
Fibers were prepared using 100 Da dextran at different concentrations. Figure shows
the effects of 100 kDa dextran concentration on the morphologies of dextran fibers. At the
lowest concentration (0.3 g/mL solvent), mainly beads with an average diameter of 800 nm
and few short fibers were formed (Fig. 11A). This indicated there was insufficient polymer
chain entanglement as shown by the Newtonian flow behavior, as indicated by the shear
stress-strain behavior or the flow behavior index of the solution as summarized in Table 8.
Increasing the polymer concentration to 0.45 g mL solvent noticeably decreased bead
formation (Fig. 1IB). 'The morphology became more fiber-like with an average diameter of
about 90 nm. Occasionally, short fibers were observed indicating that polymer chain
entanglement was still too low to produce continuous fibers. Smooth and continuous fibers
were formed when the dextran concentration was 0.6 g/mL solvent (Fig. 1IC). The average
fiber's diameter was found to be about 180 nm. Further increase in dextran concentration to
0.7 g/mL solvent resulted in a large increase in the average fiber's diameter to about 300
nm. (See Figs. 11E-H). These data establish that, for 100 kDa dextran, a concentration of
0.6 g/mL solvent is optimum.
a . Dextran-WPI fibers
Whey protein isolate was mixed into 0.6 g/mL dextran solution a t different mixing ratios
(see Table 8). Figure 12 shows the effect of whey protein isolate content on the
morphologies of electrospun fibers. The average fiber diameter slightly decreased from 192
nm (Figure 2A and F) to 6 nm (Figure 12B and G) when the mixing ratio between
dextran and whey protein content changed from 1:0 to 0.8:0.2. Further increasing whey
protein content by changing the mixing ratio to 0.75:0.25 decreased the average fiber
diameter to about 6 1 nm (Figure C and FT).
[135] The decreased fiber diameter is due to an increase in the electrical conductivity and a
decrease in the viscosity of the mixture when the whey protein isolate content was higher,
which favors stretching of the polymer jets during electrospinning, resulting in thinner
fibers.
[136] However, when the whey protein content was equal to or higher than 33%, beads on fibers
were observed (See Figs. 2D and 121, and 12E and 121), indicating that the viscosity of
the mixtures was too low. Accordingly, a whey protein content of less than 33% is
optimal.
[137] EXAMPLE 7
Analysis of Fiber Protein Content by Fourier Transform infrared Spectrometer (FTIR)
[138] FTIR was performed as described in Example . IR spectra of the mixtures having different
amount of dextran (powder) and whey protein isolate (powder) were collected (Figure
13A). The peak absorbance in the amide hand II region at around 1520 cm 1 increased
remarkably with protein content and was used as an indicator of the protein content in the
sample. The ratio between the peak absorbance at the wavenumber of around 1520 cm 1
and 1000 cm was calculated from each spectrum and the data was re-plotted in Figure
13B. interestingly, a linear relationship between the protein content (·;·;·;· and the ratio
between two peak absorbance (p,-) was found as (R -94.00%): p r ==7834.0= c . (Equation
2.2).
|I39| The relationship remained linear up to protein concentrations of 30% wt. When the whey
protein content reached 50% a non-linear relationship was observed. However, since the
target value of protein content used in this study was about 25%wt, equation 2.2 was still
valid for the quantitative analysis.
Table 9 shows the values of protein content in electrospun films prepared from mixtures of
dextran-whey protein isolate at different mixing ratio. The calculated value was not much
different from the mixing ratio, indicating that polysaccharide and protein were
simultaneously electrospun This indicated that both protein and polysaccharide were
simultaneously present m the electrospun fibers at concentrations desirable tor subsequent
conjugate reaction formation.
Table 9 Analysis of ey protein isolate content in electrospun films prepared from
dextran-whey protein isolate at different mixing ratio
] The values reported are the average value standard deviation of duplicate measurement.
] EXAMPLE 8
Analysis of WPI-dextran Conjugate Formation
] Although the WP protein was associated with the dextran fibers, we needed to confirm
that conjugation; i.e. formation of covalent bonds, was occurring. Accordingly, we
investigated the conditions where conjugates between WPI and dextran are formed,
including dextran molecular weight, humidity during annealing an mixing ratio between
WPI and dextran on WPI-dextran conjugates formation were studied.
] Gel electrophoresis was employed to verif the presence of glycoproteins in annealed
electrospun films and a UV-VI8-NIR spectrophotometer was used to examine color
development during annealing and to obtain N R spectra of electrospun films.
] EXAMPLE 9
Appearance of Heat Treated Films
[14 7 Figure 14 shows the appearance of as-spun and annealed electrospun films. The color of asspun
film was white with lower densities while annealed samples increased in density and
were slightly more yellowish (Figure 14). There was no smell detected after annealing,
indicating that no short molecular weight Maillard reaction products were formed. The
color of the films became more yellow with increasing annealing times.
[148] EXAMPLE 10
Analysis of Conjugate Formation
[149] Initial approaches to detecting glycosylated proteins by measuring the IR spectra were
unsuccessful. We expected a significant change in the absorbance of WPI-dextran IR
spectra at around 1630-1640 cm 1 due to =C stretching (Schiff-base product formation
during Maillard reaction) [16, 17]. However, R spectra obtained from mixture of WPIdextran
powder and electrospun films that were annealed at 60 C and 74 % were not
that much different. See Figure 15. This might be because the technique was not sensitive
enough to detect the presence of WPI-dextran conjugates in annealed electrospun films
compared to the presence of unreacted dextran and WPL
[150] Therefore conjugation was assessed using gel electrophoresis, as described in Example 1,
which is more effective in detecting glycoprotein formation in the samples.
[151] We selected 40 kDa dextran because it was reported that smaller molecular weight dextrans
may facilitate formation of WPI-dextran conjugates better than higher molecular weight
ones [1], allowing us to validate the method before using the target dextran (100 kDa).
Figure 16 shows the SDS-PAGE pattern of WPI-dextran-40 kDa conjugates that were
annealed at 60 "C for up to 48 hours. Unexpectedly, it seemed that WPI-dextran 40 kDa
conjugates formed in the sample that had been annealed as little as 2 hours (Lane 3).
[152] After glycosylation had been confirmed for fibers of dextran-40 kDa and WP , films were
electrospun from a blend of WPI and dextran-100 kDa. Films were annealed and sample
solutions were prepared using the same conditions as in the WPI-dextran-40 kDa samples.
The prepared samples were then subjected to a gel electrophoresis experiment n the same
gel, samples prepared from the previously annealed WPI-dextran-40 kDa eiectrospun films
were also loaded in order to compare them to solutions prepared from annealed WPIdextran-
G kDa. SDS-PAGE patterns of WPI-dextran conjugates are shown in Figure 17.
Figure 7A and 17B show images of protein-stained and glycoprotein-stained gels,
respectively. Both gels were run at the same time.
For samples prepared from dextra.n-40 kDa, the results were similar to those shown in
Figure 6 . Bands of WP were significantly diminished after the film had been annealed
for 8 hours (lane 3). With longer annealing time (lane 4 and 5 : 16 and 24 hours,
respectively), the band with larger molecular mass compounds became more intense. This
indicated that glycosylation of protein and dextran increased with longer annealing times
(Figure 17B).
SDS-PAGE patterns of eiectrospun film prepared from dextran- 00 kDa. (lanes 6-9)
indicated a similar behavior as those prepared from dextra.n-40 kDa (lanes 2-5), but with
less intensity and a higher location on the gel. We suspect the larger size of dextran- 100
kDa reduced the mobility of molecules or decreased the number of contact points between
the molecules (both factors that facilitate the reaction). Thus the reaction apparently was a
less efficient than that with the smaller dextran-40 kDa. Nevetheless, from Figure 17B, it is
apparent that conjugates are formed and th at their size was much larger than that of WPIdextran-
40 kDa conjugates.
Figure 18A and B shows SDS-PAGE profiles of WPI-Dextran (70 kDa) conjugates. As
before, the glycoprotein band became more intense with longer annealing time. The band
intensity was less than those prepared from dextran-40 kDa but more than those prepared
from dextran- 100 kDa. Standard protein ladder was also loaded into the same gel in order
to make a molecular weight standard curve for mini-gel by plotting the traveling distance
against known protein molecular weights (Figure 3.6). The glycoproteins found in
eiectrospun films prepared from dextran 40 kDa, 70 kDa and 100 kDa thus could be
estimated as >70 kDa, >200 kDa and >280 kDa, respectively.
[156| Figures 20A and B show the effect of humidity on glycosylation. There was much less
intensity of glycosylation hands in glycoprotein-stained gel when samples were annealed
under low humidity (0 %R and 44 %RH). Enhanced glycosylation was obtained at 74%
R (See Lanes 840).
jl57| Polymer molecules in electrospun fibers are well-entangled thus the mobility of molecules
is limited. Increasing humidity may increase chain flexibility, resulting in more reactions
and thus enhanced formation of WPI-dextran conjugates.
[158] The two main components in whey protein isolate used in this experiment, b-lactoglobulin
and a-lactaibumin have a molecular weight around 8 and 4 kDa, respectively. This is in
agreement with the four bands found at around 14, 18, 30 and 36 kDa which could be
attributed to monomers and climers of two main components in WPI (Figure 18A). The
band at around 60 kDa was assigned to bovine serum albumin (BSA) [7, 9].
[1 The presence of these major bands in all protein-stained gel meant that there unreacted
WPI remained in electrospun film even after annealing for 24 h. This implied that mixing
ratio between dextran and WPI could be adjusted by increasing dextran or decreasing WPI
in precursor solutions. Thus electrospun films were prepared from WPI and dextran 100
kDa. at different molar ratios. Figure 2 1 shows the SDS-PAGE pattern of those electrospun
films. The glycoprotein-stained gel confirmed the presences of glycoprotein bands in all
annealed electrospun films (lanes 2-4, 6-8, 10-12) but not in as-spun films (lane 1, 5 and 9).
The intensity of glycoproteins bands was in following manner: lanes 10-12 > lanes 6-8 >
lanes 2-4. However, there was not th at much difference in the intensity of small molecules
bands (b-lactoglobulm, -lactalbumin and BSA). Thus it might be possible that the
optimum molar ratio between dextran- 00 kDa and WPI could be higher than 2:1 used in
this feasibility study.
[160] EXAMPLE 11
Color Development During Annealing
To follow the color development of annealed electrospun films as a function of time,
absorbance spectra were measured using a UV-VIS-NIR spectrophotometer. Figures 22A
and 22B show the UV absorbance spectra of electrospun films prepared from dextran-40
kDa annealed at different times. The spectra show only one clear absorbance peak at
around 280 nn which was the same wavelength that used to detected protein residual in
samples [20, 1] The result shows that absorbance peaks increased with annealing time.
163] WPI concentration was kept constant in every solution sample; therefore, the increase in
absorbance may be due to larger size of glycoproteins compared to native WPI molecules.
This indicated that longer annealing time promoted more glycoprotein formation. The plot
between absorbance peak and annealing time looks similar to a sigmoid curve. At the
beginning of annealing, dextran and WPI molecules might need relaxation periods to
realign themselves thus there was just slightly an increase in glycoprotein formation (0-4
h). After this initial relaxation period, glycosylation may proceed at a faster rate from 4h to
24h). Between 24h and 48h, the rate of glycosylation slowed, likely due to a decrease in
reaction sites. Similar trends were also observed in electrospun films prepared from
dextran 70 kDa (Figure 23A and 23B).
164] In contrast, electrospun films prepared from dextran 00 kDa-WPI with a molar ratio of :2
displayed an unusual fluctuation in UV absorbance. (Figure 24A). Results from gel
electrophoresis experiments confirmed that there was an increase in glycoproteins over
time, regardless of dextran size; thus, the fluctuation may have been due to the
measurement itself. Since all solutions were prepared at the same concentration as
solutions prepared from dextran 40 kDa- and dextran 70 kDa-electrospun films, we suspect
that larger size of dextran molecules and/or glycoproteins might interfere with light
absorbance.
165] Solutions were therefore prepared a t lower concentration, from 5 to 3 nig/mL. It seemed
that the interference effect of molecule size on light absorbance was reduced. Figure 24B
shows a similar trend to Figure 22B and 23B. However, when the molar ratio between
dextran- OOkDa and WPI increased to 1: the interference effect returned (Figure 25A).
And increasing the molar ratio to 2:1 resulted in more interference (Figure 25B). High
levels of noise were observed in every measurement (Figure 26). These results were in
contrast with gel electrophoresis results or even simple visual observations (Figure 27), that
showed formation of colored product.
[166| EXAMPLE 12
Colorimeter Analysis
Annealed electrospun film containing WPI-dextran fibers was prepared and annealed as
described above. A colorimeter was used to assess color development of annealed
electrospun films those produced from dextran 100 kDa. As expected, the lightness of the
sample decreased while yellowness increased with annealing time (Figure 28A-C). The
less color, e.g. less yellow or brown, is better. Ideally the films are colorless. The reactions
sould be stopped after the Schiff reaction to avoid color.
[168| EXAMPLE 3
R spectra Analysis
j 9] N R spectra of electrospun films prepared fro WP and dextran 00 kDa and annealed at
different time were collected (Figure 29A and B). We did not detect a new absorbance peak
in a range of 2000 to 2300 nm, which were assigned to secondary structures of protein
molecules [20, 22]. This might indicate that there was no substantial change in protein s
secondary structures during annealing at 60 °C. This result agreed with the FT R spectra of
as-spun and annealed electrospun films (Figure 15). A peak observed located around 1400
nm which could be attributed to first overtone of O-H stretching due to the presence of
water during annealing in high humidity environment and -Fi stretching of protein
molecules [20, 23]. Overall, reflectance of annealed electrospun films shifted lower. This
was because annealed electrospun films became slightly lower in lightness with longer
annealing time; resulted in higher ability to absorb light. All spectra of annealed
electrospun films show lower reflectance values (or the higher absorbance) at around 280
nm compare to as-spun films. Generally, the darker the film, the more problematic the
film and it is difficult to achieve FTSR analysis.
As another example, each of the features of the aforementioned illustrative examples may
be utilized alone or in combination or subcombination with elements of the other examples.
For example, any of the above described systems and methods or parts thereof may be
combined with the other methods and systems or parts thereof described above. For
example, one of ordinary skill in the art will appreciate that the steps illustrated in the
illustrative figures may be performed in other than the recited order, and that one or more
steps illustrated may be optional in accordance with aspects of the disclosure. It will also
be appreciated and understood that modifications may be made without departing from the
true spirit and scope of the present disclosure. The description is thus to be regarded as
illustrative instead of restrictive on the present disclosure.
What is claimed is:
1. A method of preparing a carbohydrate-protein fiber or particle via electro-spinning
comprising steps of
preparing an aqueous solution comprising a carbohydrate and a protein,
applying a voltage of 15 to 25 kV to the solution,
collecting the fiber on a collecting plate.
2. The method of claim 1 wherein the electro-spinning is needleless.
3. The method of claim 1 wherein the carbohydrate has an aldehyde group or forms an
aldehyde group through isomerism.
4. The method of claim 1 wherein the carbohydrate is a dextran.
5. The method of claim 4 wherein the dextran molecular weight is between about 0 kDa and
about 500 kDa.
6. The method of claim 4 wherein the dextran is present at concentration of 0. g/mL to about
5.0 g/mL
7. The method of claim 1 wherein the protein is selected from microbial, animal, dairy, and
vegetable.
8. The method of claim 1 wherein the protein is a whey protein isolate (WPI).
9. The method of claim 1 wherein the aqueous solution comprises carbohydrate and protein at
a molar ratio (w w) from 50: to :50.
10. The method of claim 1 wherein the aqueous solution comprises carbohydrate and protein at
a molar ratio (w/w) from 3 : to 1:10.
11. The method of claim 1 wherein the aqueous solution comprises dextran and VVPI.
12. The method of claim 1 further comprising the step of incubating the fiber at a relative
humidity of at least 45%, for up to 24 hours, whereby a conjugated film is formed.
13. The method of claim 2 wherein the relative humidity is between 65% and 75%.
14 . The method of claim 12 wherein the temperature is in the range of 10-70 C.
15. The method of claim wherein the fiber diameter is about 00 nm to about 500 nm.
16. The method of claim 5 wherein the fiber diameter is about 50 nm to about 250 nm.
17. A method of preparing a po!ysaccharide-protem fiber by electro-spinning comprising steps
of
preparing an aqueous solution comprising 100 kDa dextran and a whey protein
isolate, wherein the dextran and the whey protein isolate are present in a molar ratio (w/w)
between 3:1 and 10:1,
applying a voltage of 15 to 25 kV to the solution whereby a fiber is created,
collecting the fiber on a collecting plate, and
incubating the fiber at a relative humidity of at least 45% for between 4 and 24
hours, w ere y a conjugated film is formed.
18. The method of claim 7 wherein the relative humidity is between 65% and 75%.
19. The method of claim 17 wherein the incubation is between 4 and 8 hours.
20. The method of claim 57 wherein the temperature is in the range of 10-70 °C.
21 . The method of claim 7 wherein the electro-spinning is needleless.