Effect of enzymatic modification on the structure and rheological properties of diluted alkali-soluble pectin fraction rich in RG-I

Abstract

This study focuses on pectin covalently linked in cell walls from two sources, apples and carrots, that was extracted using diluted alkali, and it describes changes in the rheological properties of diluted alkali-soluble pectin (DASP) due to enzymatic treatment. Given DASP’s richness of rhamnogalacturonan I (RG-I), RG-I acetyl esterase (RGAE), rhamnogalacturonan endolyase (RGL), and arabinofuranosidase (ABF) were employed in various combinations for targeted degradation of RG-I pectin chains. Enzymatic degradations were followed by structural studies of pectin molecules using atomic force microscopy (AFM) as well as measurements of rheological and spectral properties. AFM imaging revealed a significant increase in the length of branched molecules after incubation with ABF, suggesting that arabinose side chains limit RG-I aggregation. Structural modifications were confirmed by changes in the intensity of bands in the pectin fingerprint and anomeric region on Fourier transform infrared spectra. ABF treatment led to a decrease in the stability of pectic gels, while the simultaneous use of ABF, RGAE, and RGL enzymes did not increase the degree of aggregation compared to the control sample. These findings suggest that the association of pectin chains within the DASP fraction may rely significantly on intermolecular interactions. Two mechanisms are proposed, which involve side chains as short-range attachment points or an extended linear homogalacturonan conformation favoring inter-chain interactions over self-association.

Full text

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Eect of enzymatic modication
on the structure and rheological
properties of diluted alkali‑soluble
pectin fraction rich in RG‑I
Adrianna Kaczmarska , Piotr M. Pieczywek , Justyna Cybulska & Artur Zdunek
*
This study focuses on pectin covalently linked in cell walls from two sources, apples and carrots,
that was extracted using diluted alkali, and it describes changes in the rheological properties
of diluted alkali‑soluble pectin (DASP) due to enzymatic treatment. Given DASP’s richness of
rhamnogalacturonan I (RG‑I), RG‑I acetyl esterase (RGAE), rhamnogalacturonan endolyase (RGL), and
arabinofuranosidase (ABF) were employed in various combinations for targeted degradation of RG‑I
pectin chains. Enzymatic degradations were followed by structural studies of pectin molecules using
atomic force microscopy (AFM) as well as measurements of rheological and spectral properties. AFM
imaging revealed a signicant increase in the length of branched molecules after incubation with ABF,
suggesting that arabinose side chains limit RG‑I aggregation. Structural modications were conrmed
by changes in the intensity of bands in the pectin ngerprint and anomeric region on Fourier transform
infrared spectra. ABF treatment led to a decrease in the stability of pectic gels, while the simultaneous
use of ABF, RGAE, and RGL enzymes did not increase the degree of aggregation compared to the
control sample. These ndings suggest that the association of pectin chains within the DASP fraction
may rely signicantly on intermolecular interactions. Two mechanisms are proposed, which involve
side chains as short‑range attachment points or an extended linear homogalacturonan conformation
favoring inter‑chain interactions over self‑association.
Pectin constitutes up to 35% of plant cell walls, performs important functions in plant growth and development,
maintains cell–cell integrity, and, in the case of fruit and vegetables, determines rmness and texture1,2. Pectin is
considered to be the component providing visco-plastic properties to the load-bearing cellulose–hemicellulose
network in the cell wall; therefore, it plays an important role in plant cell wall rheology3. Pectin is also important
for the food industry due to its ability to increase viscosity and bind water. Moreover, pectin gelling activity
may be tuned by various parameters, such as the structure and concentration of pectin, pH, temperature, and
presence of cations4,5.
ere are three main pectic domains. Homogalacturonan (HG) consists of a linear chain of α-(1,4)-linked
D-galacturonic acid (GalA) and is known as pectin’s “smooth” region. Rhamnogalacturonans belong to the so-
called “hairy” region. e backbone of rhamnogalacturonan I (RG-I) is made of the diglycosyl repeating unit
[→ 4-α--GalpA-(1 → 2)-α--Rhap-(1 →]. Predominantly, a large proportion of rhamnose units are substituted
at O-4 with side chains composed principally of arabinans, galactans, and/or arabinogalactans1.
Neutral side chain loss and the rearrangement of their associations within RG-I are some of the most pro-
nounced and earliest changes in pectin structure during maturation, ripening, and storage in fruits and vegeta-
bles. e majority of structural changes are associated with β-galactosidase and α-L-arabinofuranosidase (ABF)
enzymatic activity, which is observed during ripening, especially for rmly bound polymers extracted by sodium
carbonate6. e percentage of methyl-esteried GalA units within the HG substructure is dened as the degree
of methyl esterication (DM), while the percentage of O-acetylated GalA units is the degree of acetylation (DA).
e numbers of methyl and acetyl groups in pectin chains aect the gelling conditions and the viscosity of pectin
solutions; thus, they are some of the major factors determining the functionality of pectin chains. e DM and
DA are strongly inuenced by the plant source as well as the extraction method. For low-methylated (LM) pectin
(DM < 50%), gelation occurs at acidic pH (2–6) and in the presence of divalent ions such as Ca2+, while high-
methylated (HM) pectin (DM > 50%) form gels in the presence of greater than 55% sugar or a similar co-solute
OPEN
Institute of Agrophysics, Polish Academy of Sciences, Doświadczalna 4, 20-270 Lublin, Poland. *email: a.zdunek@
ipan.lublin.pl
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at pH < 3.57. Hydrogen bonds and electrostatic interactions play a crucial role in the gelation mechanism of
HM pectin, while for LM pectin the “egg-box” model describes binding processes and junction zone formation
between non-esteried GalA units and calcium ions8. Characteristics of the pectin polymer backbone, including
its intrinsic exibility or stiness, play a major role in the rheological properties in solution and inuence the
order/disorder state of the system on a supramolecular scale, especially while dierent levels of chain association
may be involved in network formation9.
e diluted alkali-soluble pectin (DASP) fraction of the cell wall pectic matrix extracted with sodium carbon-
ate is considered to be the covalently linked fraction of the cell wall. As previously reported, these molecules show
the distinctive feature of creating a self-assembled network on mica10. Atomic force microscopy (AFM) imaging
and coarse grain simulations have conrmed that the network-like appearance on mica originates from rhamnose
units separating two sections of HG and the creation of kinks at the characteristic angle of 118°11. Previous studies
have demonstrated that DASP from fruits like pear or apple shows gelling ability dependent on concentration,
pH, or monovalent and divalent cations in aqueous medium, indicating the possible application of this polysac-
charide fraction due to its low methylation level12. However, due to the signicantly dierent conformations of
RG-I and HG2, we hypothesize that the rheology of DASP, which is rich in RG-I13, may be largely aected by the
side chains and the presence of the kinks caused by rhamnose interspaced with GalA.
e goal of this paper was structural characterization at the supramolecular scale and investigation rheological
properties of the DASP fractions extracted from two horticultural sources (apple and carrot) and structurally
changed by enzymatic modication. In this experiment, RG-I acetyl esterase (RGAE), rhamnogalacturonan
endolyase (RGL), and ABF were used. RGAE is an enzyme that participates in the deacetylation of GalA in
the RG-I backbone. RGL participates in the endotype eliminative cleavage of L-α-rhamnopyranosyl-1,4-α-D-
galactopyranosyluronic acid bonds of RG-I domains in ramied hairy regions of pectin, leaving L-rhamno-
pyranose at the reducing end and 4-deoxy-4,5-unsaturated D-galactopyranosyluronic acid at the non-reducing
end14,15. ABF preferentially removes α-1,2- and α-1,3-linked arabinose from side chains of either arabinan or
arabinoxylan, and it hydrolyses α-1,5-linked arabino-oligosaccharides at a low rate14. It is hypothesized that
the detachment of the rhamnose and side chains aects pectin rheology. In this study, the eect of selective
modication of pectin chains was compared for apple and carrot DASP and studied by high-performance liquid
chromatography (HPLC), Fourier transform infrared (FTIR) spectroscopy, AFM imaging analysis, and rheologi-
cal measurements of pectin aqueous solution.
Results and discussion
Nanostructure
Figure1 shows representative AFM height images of DASP from apple and carrot in the control (buer)
batch and aer 120min of incubation in three enzymatic cocktails: E1 (RGL + RGAE), E2 (ABF), and E3
(RGL + RGAE + ABF). Zoomed regions of the DASP incubated in the buer presented in Fig.1a,f show bers
only in the form of rod-like structures with linear segments of variable length and separated by bending or
branching points. Such features have been previously observed for DASP fractions extracted from apple, carrot,
or pear10,16 and have been explained as the result of rhamnose interspersion with GalA11. A similar structure was
created on the mica by the DASP bers aer applying the E1 treatment (Fig.1b,g). In the sample treated with
E2 (Fig.1c,h), larger aggregates and longer chains were observed and were particularly pronounced for carrot
(Fig.1h). Additionally, with both the E2 treatment and the combination of the three enzymes (E3) (Fig.1d,i),
short chains and small molecules were mostly noted.
According to the image analysis performed on the AFM scans, the observed structures were categorized as
“hairy” molecules or “smooth” molecules. Additionally, the total length of the branched molecule was calculated
as the sum of lengths of the branches belonging to the molecule. e average total lengths of the molecules clas-
sied as hairy, before being placed in buer, were approximately 585 ± 23nm and 443 ± 23nm for apple DASP
(DASP-A) and carrot (DASP-C), respectively (Fig.2a). is length is consistent with that previously obtained
by another study17, which was from 20 to 1000nm for the sodium carbonate pectin fraction extracted from dif-
ferent fruits. Contour lengths of alkali-treated sugar beet pectin were in the range of 20–520 nm18, similar to the
molecule length for Na2CO3 extracts obtained from mature green tomato fruits (20–400nm)19.
Aer placing the pectin in the buer, the lengths of hairy molecules (Fig.2a) did not change signicantly
for apple, while they increased for carrot. e length of molecules classied as smooth, before being placed in
buer (Fig.2b), was shorter than 100nm, and the incubation with buer for 120min caused apparent shortening
for apple and a slight increase for carrot (Fig.2b). e eect of incubation with buer, reected by changes in
parameters aer 120min of incubation, suggests that pectin incubated in buer alone may undergo structural
changes leading to self-aggregation. Since pectin was incubated in buer at pH 7, this result may be similar to
that previously obtained for DASP-A and may be explained by the mechanism of high electrostatic repulsion
between fully dissociated macromolecules that probably blocked the formation of extended pectin chains4.
Enzymatic treatment with cocktails E1 and E3 did not cause a statistically signicant change in the length
of the hairy molecules extracted from apple (Fig.2a). In the case of carrot (Fig.2a), treatment with E1 caused
a slight but non-signicant increase in length, while treatment in E3 caused a statistically signicant decrease
compared to incubation with buer. Analysis of the number of segments (Fig.2d) and the average length of
segments (Fig.2c) revealed that the total length of hairy molecules was related to the number of segments but
that, simultaneously, the segments seemed to become slightly shorter aer incubation with enzymes. e most
pronounced eect on the structure of DASP in both materials was obtained when E2 treatment was applied
(Fig.2a). ABF was the only active enzyme in the E2 treatment and caused a signicant increase in the total length
of hairy molecules (up to almost 1.5µm aer 120min). is eect was clearly associated with an increase in the
number of segments and their only slight shortening during incubation (Fig.2d). It is also worth noting that
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the chains classied as smooth (Fig.2b) had lengths similar to those of segments of hairy molecules (Fig.2b),
i.e., less than 100nm. e smooth molecules were unaected by enzymatic treatment even with E2 (Fig.2b). As
ABF preferentially removes α-1,2- and α-1,3-linked arabinose from side chains, the eect of E2 on the structure
of pectin molecules could be explained by the gradual removal of arabinose, followed by aggregation of RG-I
molecules, thereby resulting in a three-fold increase in the number of branches per molecule (from about ve
to 15 segments per molecule), as shown in Fig.2d.
Figure1. AFM height images of control (buer) and enzymatically modied (incubation time 120min) apple
DASP (a–d) and carrot DASP (e–h) on mica. Scale bar represents 1µm. Illustrations show two main categories
of structures detected on topological AFM images: hairy and smooth molecules.
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Contrary to the application of ABF alone, the simultaneous action of ABF with RGAE and RGL enzymes
(E3 treatment) did not result in aggregation and did not increase the lengths of hairy molecules. e lengths of
hairy molecules aer incubation in E3 were 576 ± 26nm and 356 ± 12nm for DASP-A and DASP-C, respectively.
A decrease in the number of branches per molecule, from seven side branches for both sources to six for apple
pectin and ve for carrot pectin, was also observed for this treatment. Shortening of hairy molecules and a
decrease in the number of branches due to E3 were more pronounced for DASP-C than for DASP-A. is higher
fragmentation of the carrot pectin chain may have resulted from the greater RG-I content (62.90mol%) than in
apple (41.50mol%), as was previously described13, which provides more sites of action for pectinolytic enzymes.
Incubation in the E1 enzyme mixture did not cause signicant dierences in branch lengths (Fig.2c) for either
source. It is suspected that the function of the RGL enzyme may have been impaired because the abundance of
arabinose side chains prevented access to the chain due to steric hindrance.
A slight decrease in segment length (Fig.2c), which could be attributed to the lengths of side branches, was
noted for the E2 treatment. Moreover, for the combination of the three enzymes (E3), a decrease in the average
length of smooth molecules (Fig.2b) was also observed, indicating chain fragmentation. is may suggest that
the simultaneous action of the enzymes modifying the RG-I skeleton and the enzymes that remove the arab-
inose side chains allowed for more eective fragmentation of the pectin chains. Hence, this result supports the
above explanation that the arabinose abundant in large amounts in the studied fraction could limit the access of
enzymes that modify the RG-I backbone.
Functional groups
e FTIR spectra obtained for DASP-A and DASP-C, both native and treated with E3 for 120min, are shown in
Fig.3. e overall shape of a polysaccharide spectrum is determined by the polysaccharide composition of the
backbone but can also be strongly inuenced by the side chain constituents20. e wavelength and intensity of
the bands allow the evaluation of possible changes in polysaccharide composition. For all samples, characteristic
absorption regions can be distinguished. e shape of each spectrum had a similar pattern, which is characteristic
of DASP polysaccharides2,21,22.
For all samples, an absence of a band in the range of 1745–1700 cm−1, which is related to the vibration of
esteried groups, suggests a lack of esters in the studied pectin fractions. is was quantied using HPLC
measurements, and no methyl groups were detected for native pectin from either source. is was probably
caused by the de-esterication of the pectin during sodium carbonate extraction16. e DA values determined
by HPLC were 5.59% and 7.48% for native apple and carrot, respectively. is suggests incomplete degradation
Figure2. Changes in structural parameters of DASP molecules from apple and carrot treated with various
combinations of enzymatic modication aer 120min. E1: DASP water solution with RGAE and RGL; E2:
DASP water solution with ABF; E3: DASP water solution with RGAE, RGL, and ABF. Bars represent standard
error (n is the number of replicates). Dierent letters indicate statistically signicant dierences (ANOVA,
p < 0.05).
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of the ester linkage between the acetyl group and the GalA residue in the pectin chains; however, these low
values are supported by the lack of peaks on the FTIR spectra for acetylated carbonyl groups (1730 cm−1) and
the stretching of C–O–C in acetyl ester (1250 cm−1), which is characteristic of acetylated pectic materials23. A
broad band in the FTIR spectrum of the tested pectin, in the range of approximately 1260–1200 cm−1, could
be considered the peak corresponding to C–O–C stretching; however, in this case it could also be caused by
C–O stretching vibrations in pectin, as was previously described for DASP from pear24. Nevertheless, the lower
intensity of the broad band around 1240 cm−1 for both samples treated with RGAE could be the eect of partial
de-esterication of acetyl groups.
e band around 1590 cm−1 was assigned to the asymmetric stretching of COO– in polygalacturonic acid,
representing non-esteried carboxyl groups in pectin, while the peak at approximately 1407 cm−1 represented
symmetric stretching vibrations of carboxylic anions. e band at 1330 cm−1, which was assigned to ring vibra-
tion, was present for all samples. As previously shown, the DASP fraction showed a high intensity at this peak
compared to the fractions extracted with water and imidazole18. Based on FTIR spectrum absorbencies in the
range of 1200–900 cm−1 (ngerprint region), it is possible to determine groupings that are specic to each poly-
saccharide. e main component inuencing the change in the shape of this ngerprint region due to enzymatic
treatment is GalA, which shows main absorbance regions at approximately 1140, 1090, 1070, and 1030 cm−1.
However, it has been shown that dierent pectic compounds also can show dierent characteristic positions of
maximum bands in this region21,25. e absorbance bands at approximately 1075 cm−1 and 1045 cm−1 also suggest
the presence of RG-I domains20. Changes in the intensity of bands in this region and/or the disappearance of
Figure3. Attenuated total reectance (ATR)–FTIR absorbance spectra of DASP extracted from apple and
carrot in their native states (dotted lines) and aer E3 enzymatic modication (solid lines) in the range of
1800–800 cm−1.
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peaks observed for modied fractions may suggest fragmentation changes in the DASP main chain. e peak at
about 950 cm−1, which is characteristic of RG-I21, was assigned to galactose side chains10 and did not change in
intensity for the E3 modied DASP-A. In contrast, its disappearance was observed for the enzymatically treated
DASP-C. is could be the result of rhamnose removal and, therefore, galactan side chain loss for the carrot
sample. Bands in the wavenumbers in the range of 900–800 cm−1 belong to the anomeric region and can be used
to dierentiate the α- and β-congurations of anomeric carbon. Peaks at approximately 890–850 cm−1 indicate
the presence of galactopyranose and arabinofuranose units in a sample20. For both DASP-C and DASP-A, a
disappearance of the peak at 890 cm−1 was observed for modied pectin. In addition, for DASP-A, a reduction
in the peak intensity at 850 cm−1 was observed. ese changes may suggest a rearrangement of the bonds related
to side chains in the pectin molecule.
Rheological properties
Flow curves collected aer 6% DASP-A and DASP-C solutions were treated with buer or enzymes for 120min
are shown in Fig.4. A power law (Ostwald–de Waele) and the Herschel–Bulkley uid model were tted to
the shear rate–shear stress curves for all samples. e consistency coecient (K) and ow behavior index (n)
were used to describe uid behavior (Table1). All n values were less than 1, showing that samples behaved as
pseudoplastic shear-thinning uids, as reported previously for other pectin solutions26. is indicates that their
apparent viscosity decreased with increasing shear rate and that the macromolecular network was oriented or
deformed in the direction of ow.
e E2 (ABF) treatment led to a decrease in K for both fractions, indicating a weakening of the binding of
the network. It was concluded that arabinose side chains were involved in macromolecular entanglements in the
native fractions, which resulted in higher viscosity for the pectin in buer27. e strong impact of ABF on the
structure of DASP could be caused by the relatively high content of arabinose in the tested fractions. e content
of this monosaccharide was 23.6 ± 0.1mol% in DASP-A and 19.8 ± 2.3mol% in DASP-C (TableS1). Moreover,
the tested fractions diered in rhamnose content: 3.8 ± 0.2mol% for DASP-A and 8.4 ± 1.8mol% for DASP-C.
It is worth noting that, for DASP-A, which contains greater amounts of arabinose, decreases in viscosity and
pseudoplastic character aer incubation in ABF were much more pronounced. e suggested role of arabinose
in the formation of a compact network was supported by a decrease in the yield stress (G0), which describes the
minimum shear rate needed to initiate ow of the material, of samples treated with ABF. In contrast, a strong
increase in this parameter, which was observed with the E3 treatment, indicated the formation of a dense net-
work, which was resistant to mechanical disruption, for the debranched polymer. An increase in K with the E3
treatment, combined with a decrease in the ow index, suggests a stronger pseudoplastic character of the DASP
solution aer simultaneous deacetylation and removal of arabinose and rhamnose. e DASP-C sample, aer
modication with this enzymatic cocktail, showed the highest pseudoplasticity of all tested solutions. e E3
treatment also resulted in an increase in the viscosity of both sources. is may indicate a greater possibility
of particle movement controlled by the entanglements of side chains attached to the rhamnose units as well as
acetyl groups, which can hinder the adoption of binding-favorable conformations by the polymer28. In addition,
rhamnose inclusions themselves can limit the cross-linking of chains29. An increase in the viscosity of DASP was
previously observed during storage of carrot roots26. at study hypothesized that hydrogen bonding between
smooth pectin chains and hydrophobic interactions by the methyl groups of pectin chains had occurred as a result
of the enzymatic modication naturally occurring in roots. erefore, it is suspected that E3 treatment resulted
in predominantly unbranched acid polymers. Considering the low DM as a result of extraction with sodium
carbonate, under these conditions the tendency to self-aggregate in deionized water is reduced. Hence, mol-
ecules adopt a more extended conformation that favors interactions between the chains30,31. is interpretation
Figure4. Averaged upward ow curves (shear stress vs. shear rate) for apple (a) and carrot (b) DASP solutions
treated with dierent enzymatic combinations. E1: DASP water solution with RGAE and RGL; E2: DASP water
solution with ABF; E3: DASP water solution with RGAE, RGL, and ABF.
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is supported by the recently highlighted role of intermolecular interactions in the mechanical properties of LM
pectins, whose extended conformation at neutral pH can increase the elastic character of the mixture32.
To gain insight into the viscoelastic properties of the studied solutions, oscillatory measurements were per-
formed. All the investigated samples exhibited behavior that was more solid-like than liquid-like, as evidenced
by the storage modulus G′ being much greater than the loss modulus G″ in amplitude sweep tests. is was
conrmed by values of the loss factor tan δ < 1 (G′ > G″).
A decrease in the storage modulus G′ was noted as a result of the E1 treatment for fractions from both sources.
As a result of potential depolymerization of the RG-I backbone, this process reduces the average molecular
weight of the polymer, leading to decreased entanglements and, hence, overall network strength reduction and
disruption of crosslinking. Flow point and linear viscoelasticity values slightly decreased for DASP-A, which was
a consequence of the process described; however, for DASP-C, the opposite trend was observed. e increase in
these parameters suggests that the structure of deacetylated DASP-C, which is a result of enzymatic modication
with RGAE, is more resistant to deformation of this material.
A large decrease in the storage modulus G′, which was observed aer removing the arabinose side chains
(E2 treatment), combined with an increase in the loss factor G″, indicated a signicant decrease in the elastic
properties of pectic gels. is may suggest that arabinose chains, as binding points in the pectin network, exhibit
solid-like behavior. For RG-I–enriched pectin, arabinose was involved in gel formation under cation and acid
conditions, and improved network formation and enzymatic debranching resulted in a decrease in side-chain
entanglements and, hence, looser pectin molecule conformation27. Similarly, decreases in elastic properties
and breaking force have been observed for debranched highly methylated citrus pectin gels33. is study also
showed that untreated and debranched pectin gels were governed by the same type of interactions. However,
for gels formed by less branched pectins, the network became less entangled, with fewer inter-chain connec-
tions, between the polymer molecules, which resulted in an overall decrease in elasticity. For the E2 treatment,
increases in the ow point and linear viscoelasticity limit were observed, which indicates that the system was
able to retain the molecular properties of the pectin network as the strain increased. It is worth noting that the
E2 treatment, by selectively excising the arabinose units, le the galactan side chains intact. erefore, it is pos-
sible that aggregate-stabilizing properties of galactans became apparent in this sample, as was shown by another
study34. e rheological parameters obtained may indicate that, for pectin at a concentration of 6%, molecular
association occurred with the formation of intermolecular interactions. When the E3 enzyme combination was
applied, similar viscoelastic parameters were obtained for both sources. is may indicate that the degradation
of arabinose side chains and deacetylation and removal of rhamnose chains have major eects on the observed
dierences between the DASP of these two materials.
As a result of enzymatic modication, only a small eect on intrinsic viscosity was observed for DASP-A
(E3 had the greatest impact on this source). A much more signicant eect of enzymatic treatment on intrinsic
viscosity was noted in the case of DASP-C. e observed decrease in intrinsic viscosity following enzymatic
modication suggests a signicant impact on the molecular structure, particularly in DASP-C. is reduction
in intrinsic viscosity, which was notable aer the E2 and E3 treatments, indicated the formation of compact
Table 1. Rheological properties of native and modied DASP from apple and carrot. G′, elastic (storage)
modulus; G″, viscous (loss) modulus; tan δ, loss factor; K, consistency coecient; n, ow index; G0, initial
shear stress. Dierent letters indicate statistically signicant dierences (ANOVA, p < 0.05).
Parameter Unit
Pectin source
Apple Carrot
BUFFER E1
(RGAE + RGL) E2 (ABF) E3
(RGAE + RGL + ABF) BUFFER E1
(RGAE + RGL) E2 (ABF) E3
(RGAE + RGL + ABF)
G′Pa 310.83 ± 231.30ab 1154.61 ± 139.91a32.29 ± 17.23ab 338.77 ± 145.85ab 405.53 ± 28.92b167.26 ± 154.87ab 43.67 ± 25.38ab 207.33 ± 119.49ab
G′/G″7.52 ± 3.14ab 10.74 ± 1.42b4.38 ± 0.99ab 8.34 ± 1.43ab 6.77 ± 0.51ab 6.44 ± 1.08ab 3.51 ± 0.93a 6.93 ± 2.48ab
tan δ 0.14 ± 0.00abc 0.09 ± 0.01bc 0.24 ± 0.05a0.13 ± 0.03ab0.15 ± 0.01abc 0.16 ± 0.03abc 0.30 ± 0.07c0.15 ± 0.07ab
Flow point % 6.07 ± 3.83ab 5.76 ± 1.26bc 16.09 ± 4.50ab3.77 ± 1.95a1.93 ± 0.85a12.85 ± 7.76abc 20.60 ± 10.64c3.77 ± 1.62ab
Linear vis-
coelasticity
limit %3.72 ± 1.83abc 1.79 ± 0.50bc 5.76 ± 2.20a1.54 ± 0.75a1.34 ± 0.90a3.41 ± 1.47abc 7.20 ± 2.23c1.88 ± 0.79ab
Power law model
KPas 2.59 ± 3.99a1.39 ± 1.46a0.19 ± 0.09a2.62 ± 2.76a9.33 ± 3.66ab 20.40 ± 13.57b5.42 ± 2.16a23.56 ± 12.54b
n0.56 ± 0.16bc 0.63 ± 0.19cd 0.86 ± 0.07d0.54 ± 0.13abc 0.41 ± 0.06abc 0.32 ± 0.14ab 0.49 ± 0.09abc 0.29 ± 0.08a
Herschel–Bulkley model
G0Pa 3.63 ± 5.75a3.21 ± 4.08a0.06 ± 0.11a3.71 ± 5.61a10.79 ± 6.97ab 31.17 ± 20.41bc 7.94 ± 4.57a32.98 ± 16.12c
KPas 1.07 ± 1.16abc 0.46 ± 0.25ab 0.19 ± 0.09a1.28 ± 0.9abc 4.76 ± 0.65d3.07 ± 1.68cd 2.79 ± 0.95bcd 5.08 ± 2.31d
n0.63 ± 0.12ab 0.73 ± 0.09bc 0.87 ± 0.07c0.61 ± 0.1ab 0.50 ± 0.03a0.59 ± 0.14ab 0.58 ± 0.05ab 0.48 ± 0.04a
Viscosity
(at 10 s−1)Pas 0.93 ± 0.89ab 0.65 ± 0.55a0.14 ± 0.05a1.58 ± 1.17ab 3.13 ± 0.87bc 4.91 ± 2.08c2.13 ± 0.77ab5.19 ± 1.90c
Intrinsic
viscosity mg L−1 158.33 148.53 151.56 123.29 293.37 242.31 160.28 147.51
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molecular aggregates, as shown in AFM images (Fig.1g,h). ese aggregates may signify an increase in molecular
exibility and compactness, possibly resulting from the dissociation of supramolecular aggregates in the native
fraction and the reduction of intramolecular forces30 induced by selective degradation of the pectin chain. e
dierent reactions of the apple and carrot fractions to enzymatic modication may be related to dierences in
their monosaccharide composition of initial fractions (TableS1). Since solutions lacked sucrose and cations,
apart from the low salt content of the enzyme buer, the network formed in the solution was the result of the
DASP fraction’s natural tendency to self-assemble, which was previously noted and observed with AFM at low
concentrations10. erefore, it can be expected that the association of pectin chains of the DASP fraction in
aqueous solution in the absence of cations occurs by two mechanisms. For native DASP polymers, which are
composed of both smooth and hairy structures, neutral side chains are involved in providing multiple short-
range attachment points for intermolecular entanglement, which is more favorable than electrostatic repulsion
between GalA chains. In contrast, for smooth and linear regions, which are rich in GalA at neutral pH, strongly
charged molecules cause intramolecular repulsion; thus, a more extended conformation results8. Short linear
sections with high mobility cause interactions between the chains to be more favorable than self-aggregation.
Conclusions
is study demonstrated changes in the structure and rheological properties of DASP fractions extracted from
apples and carrots under the inuence of enzymes that selectively modied the pectin backbone and side chains.
e structure of pectin in the DASP fractions had a signicant inuence on rheological properties. is was
supported by changes in the structure, chemical composition, and rheological properties of samples observed
as a result of enzymatic modication of RG-I fragments. e removal of rhamnose units, simultaneously with
the deacetylation and removal of arabinose side chains, resulted in similar rheological parameters of pectic gels
from two plant sources that were dierent from the properties of the unprocessed samples. erefore, it can be
concluded that rhamnose may be the factor determining the properties of pectin matrices in solution. Modi-
cation with ABF had the greatest impact on the properties of this pectin fraction. Arabinose, which is present
in side chains, is involved in the formation of the pectin network and aects the pseudoplastic properties and
viscosity, but not the mechanical strength, of pectin solutions. At the same time, a signicant increase in the
lengths of the chains aer the removal of arabinose indicates that the side chains are a hindrance limiting the
binding of polymer chains.
It can thus be concluded that the association of the pectin chains of DASP fractions in an aqueous solution
in the absence of cations may occur due to the crucial role of intermolecular interactions according to two
mechanisms: side chains as short-range attachment points and an extended linear HG conformation favoring
inter-chain interactions over self-association.
Materials and methods
Pectin source
e research material included apples cv. Najdared (Malus domestica Borkh.) and carrots cv. Brava (Daucus carota
subsp. sativus). Material was harvested in Poland in October 2020 and then stored in a cold room at 2°C and
normal atmosphere for 2days until preparation. Pulp was prepared from 102kg of raw apples and 34kg of raw
carrots. Both were peeled and sliced. e juice was pressed, and the remaining pomace was homogenized. en,
the prepared material was frozen at − 18°C for further analysis. Alcohol-insoluble residue (AIR) was prepared
identically for both plants, according to the method described by Renard35 with some modications. e pulp
was mixed with ~ 70% ethanol (solid–liquid ratio of 1:10, w/v) for 15–30min, and then the mixture was ltered
on a nylon lter, and the residue was stirred again with ethanol. is procedure was repeated until a negative
result of the phenol–sulfuric acid test36 was obtained, thereby conrming the absence of sugar in the pulp. Next,
the sample was washed with 96% ethanol and subsequently with acetone and then dried at 45°C.
DASP extraction
Sequential extraction was performed for both sources according to the method proposed by Redgwell and
Selvendran37 with certain modications. AIR was stirred in deionized water (solid–liquid ratio of 1:9, w/v) for
24h at 21°C and then centrifuged (5000rpm). Supernatant was collected as a water-soluble pectin fraction, and
the sediment was mixed with 0.1M cyclohexane-trans-1.2-diamine tetra-acetate (CDTA, pH 6.5) and stirred
at 21°C for 24h. Aer centrifugation, the supernatant was separated as a chelate-soluble pectin fraction, and
0.05M sodium carbonate (Na2CO3), with the addition of 20mM sodium borohydride (NaBH4), was added to
the residue and stirred for 24h at 21°C. e DASP fraction was collected aer centrifugation as a supernatant
and encoded as DASP-A or DASP-C, for apple-extracted or carrot-extracted samples, respectively. e DASP
fraction was dialyzed in an open system using ZelluTrans/ROTH® membranes (Carl Roth GmbH & Co. KG,
Germany; MWCO 3500Da), and then crude extract was lyophilized. DASP from apple and carrot was extracted
with a yield of 0.44% and 0.61% of fresh weight (25.58 and 21.30% of dry weight), respectively13. e chemical
composition of DASP from both sources is presented in TableS1 (Supplementary Information).
Determination of DA and DM
To determine the DM and DA of pectin, samples were saponied with 0.2M NaOH to produce methanol and ace-
tic acid, which were then measured by HPLC (C18 column, Bionacom velocity LPH-C18, 300Å, 4.6 × 250mm,
5 microns, RI detector). e method of Levigne etal.38 with some modications by Yu etal.39 was used. Pectin
samples of 5mg were suspended in 0.5mL 0.2M NaOH and incubated at 4°C for 120min. en, the mixture
was neutralized with 0.5mL 0.2M H2SO4, centrifuged for 10min, ltered through a 0.22µm syringe lter,
and injected into the HPLC column (injection volume 20µL, mobile phase 4mM sulfuric acid at a ow rate
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of 0.8mL min−1). Standard solutions of methanol and acetic acid were prepared and analyzed under the same
conditions. e analysis was performed in triplicate.
Enzymatic treatment
e DASP fractions from apples and carrots were treated with enzymes that degrade the RG-I backbone and its
side chains. ree types of enzymes were used: RGAE (BtRme NC (CE NC), BT4158, E.C. 3.1.1), RGL (BtRge9A
(PL9), BT4183, E.C. 4.2.2.23), and ABF (CjAbf51B (GH51), E.C. 3.2.1.55). All enzymes were purchased from
NZYTech and provided in 35mM Na–HEPES buer (pH 7.5, 750mM NaCl, 200mM imidazole, 3.5mM CaCl2,
and 25% v/v glycerol).
e quantities of enzymes were selected on the basis of their activity15,40,41 and the chemical composition of
DASP with 10% excess. Per 1mg of DASP, 0.22 U of RGL, 9.9 U of RGAE, and 2.1 U of ABF were used. Volumes
were obtained according to protein concentrations (RGAE: 0.5mg mL−1; RGL: 0.5mg mL−1; ABF: 0.25mg mL−1).
DASP water solutions were incubated in three dierent enzyme cocktails (Table2). As a control (no enzymatic
treatment), the same buer solution in which the enzymes were delivered was added to DASP water solutions.
e pH values of all tested solutions were in the range of 7.30–7.60. Control and enzyme-treated samples
were incubated in a water bath at 37°C for 120min. Aer incubation, the samples were cooled in an ice bath for
5min to stop the enzymatic reactions, mixed, and used for further analysis.
AFM imaging and analysis
Aer enzymatic modication, 30 L of each DASP sample, with a concentration of 0.02mg mL−1, was distributed
on freshly cleaved mica sheets (EMS, Hateld, PA, USA) using a POLOS SPIN150i-NPP spin coater (SPS-Europe
B.V., Putten, the Netherlands). Samples were observed (aer drying in a desiccator at 22°C overnight) using
a Multimode 8 AFM with a Nanoscope V controller (Bruker, Billerica, MA, USA), a SCANASYST-AIR-HR
cantilever (Bruker, Billerica, MA, USA), and a nominal spring constant of 0.4 N m−1. e observations were
conducted in ambient air at room temperature. e following scan settings were applied: scan size of 4 × 4m
with a resolution of 1024 × 1024 points and a scan rate of 3.91Hz. In total, at least 10 images of each sample
representing dierent regions of the mica sheets were collected. Preliminary processing of images was conducted
using Gwyddion 2.52 42. Geometrical features of DASP structure were calculated with a MATLAB R2011a script
(MathWorks, Natick, MA, USA). Molecules visible on the AFM images were classied as hairy or smooth,
according to the presence of branch points in the chains (Fig.1). A single segment was dened as the section
between the nearest branching points or between a branching point and the end of the molecule. e total length
of hairy molecules (as the sum of their segments), the length of smooth molecules, and the length of a single
segment were determined. Moreover, the average number of branches (segments) per molecule was determined.
FTIR spectroscopy
DASP was dissolved in deionized water at a concentration of 6% (m/v). Aer preparation, samples were vor-
texed (3000rpm) and then mixed overnight. DASP was treated with mixture of E1, E2, or E3 and incubated as
described the “Enzymatic treatment” section. Subsequently, samples were cooled in ice for 5min, mixed over-
night, and freeze-dried. FTIR spectra were collected using a Nicolet 6700 FTIR (ermo Scientic, Madison,
WI, USA) with the Smart iTR attenuated total reectance (ATR) sampling accessory. All samples from both
sources were analyzed under the same conditions. Spectra were collected in the range 4000–650 cm−1 with a
spectral resolution of 4 cm−1. Measurements were performed in three repetitions with 200 scans averaged for
each repetition. e baseline corrections were performed using OMNIC soware (ermo Scientic). e nal
average spectrum was calculated from collected data and normalized to 1.0 at 1019 cm−1 using OriginPro 8.5
soware (OriginLab Corporation, Northampton, MA, USA).
Determination of rheological properties
DASP 6% (m/v) solutions were prepared in the same way as for FTIR analysis (“FTIR spectroscopy” section);
treated with a mixture of E1, E2, or E3; and incubated as described in the “Enzymatic treatment” section. Subse-
quently, samples were cooled in ice for 5min. Aer cooling, samples were stabilized at room temperature before
the oscillatory test and ow behavior measurements were performed.
For intrinsic viscosity measurements, a stock solution of 20mg mL−1 DASP in deionized water was prepared.
A mixture of enzymes (E1, E2, or E3) was added to the well-dissolved sample. Aer 120min of incubation, the
sample was cooled in ice. From the stock solution, dilutions (5–18mg mL−1) were prepared to obtain intrinsic
viscosity curves.
Table 2. Code list and description of the treatments used.
Treatment code Description
E1 RGAE (9.9 U mg−1) + RGL (0.22 U mg−1)
E2 ABF (2.1 U mg−1)
E3 RGAE (9.9 U mg−1) + RGL (0.22 U mg−1) + ABF (2.1 U mg−1)
BUFFER 35mM Na–HEPES buer, pH 7.5, 750mM NaCl, 200mM imidazole, 3.5mM CaCl2, and 25% (v/v) glycerol
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Rheological measurements were performed at 20°C using a Discovery Hybrid Rheometer (HR-1) by TA
Instruments (New Castle, PA, USA) with a cone plate sensor (40mm diameter and 2.007° angle) with a 0.56mm
gap between the cone apex and the plate.
Viscoelastic properties
e oscillatory test was conducted with amplitude sweeps to describe the storage (G′) and loss (G″) moduli
of the obtained networks. Measurements were performed while keeping the frequency at a constant value of
0.5Hz and using a logarithmic sweep with strain in the range of 0.1–50% (25 points per decade; volume of the
deposited sample 1mL).
Flow behavior
Shear stress vs. shear rate dependences (ow curves) were measured between shear rates of 10–600 s−1 and
600–10 s−1 (logarithmic sweep, 15 points per decade). e viscosity was recorded at constant shear rate of 10 s−1.
A power law model (Ostwald–de Waele model) and the Herschel–Bulkley model were applied to obtain ow
curves in order to determine the rheological behavior of samples.
e power law model is given as Eq.(1):
where
σ
= shear stress, K = consistency index (Pa sn), γ = shear rate (s−1), and n = ow behavior index.
e Herschel–Bulkley model is described by Eq.(2):
where σ = shear stress, σ0 = initial shear stress (Pa), K = consistency index (Pa sn), γ = shear rate (s−1), and n = ow
behavior index.
Intrinsic viscosity
e variable shear rate of 10–400 s−1 and 400–10 s−1 (logarithmic sweep; 25 points per decade) was used to
measure viscosity of the solutions with a volume of the deposited sample of 0.80mL. e intrinsic viscosities of
samples were determined via an extrapolation of Eq.(3) to a concentration c equal to 043:
where ηsp = the specic viscosity that can be obtained from the relative viscosity (η solution/ηsolvent), c = polymer
solution concentration, and kH = the Huggins constant.
Statistical analysis
e data were analyzed with multi-way ANOVA and a post hoc Tukey honestly signicant dierence test (sig-
nicance level p < 0.05) using the “stats” package (version 4.1.2) of R (R Core Team, 2013) and Statistica 13.1
(StatSo, Krakow, Poland).
Ethical approval
All methods were in accordance with the International Union for Conservation of Nature Policy Statement on
Research Involving Species at Risk of Extinction and the Convention on International Trade in Endangered
Species of Wild Fauna and Flora. e botanical material was harvested from a commercial orchard located
in Ostrowiec (52°9′59.60′′ N, 20° 3′ 23.84′′ E) and an agricultural experiment station located in Dębowa Góra
(51°51′8.38′′ N, 20°7′1.76′′ E), Poland.
Data availability
e datasets generated during and/or analyzed during the current study are available from the corresponding
author on reasonable request.
Received: 21 December 2023; Accepted: 14 May 2024
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(1)
σ
=
Kγn
(2)
σ
=
σ0
+
Kγn
(3)
ηsp
c
=[η]+kH[η]2
c
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Acknowledgements
is work was supported by the National Science Centre, Poland (grant number 2019/35/O/NZ9/01387).
Author contributions
A.K.: Writing—original dra, Conceptualization, Methodology, Formal analysis, Visualization, Investigation.
P.M.P: Writing—review and editing, Methodology, Data curation, Conceptualization, Visualization, Validation,
Formal analysis. J.C.: Writing—review and editing, Methodology, Investigation, Formal analysis. A.Z.: Writ-
ing—review and editing, Resources, Data curation, Supervision, Project administration, Funding acquisition.
All authors have read and agreed to the published version of the manuscript.
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Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
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