Ijbiomac D 23 13059 r1 Reviewer

International Journal of Biological Macromolecules
Effects of pectin structure on carotenoid bioaccessibility in simulated juice model

--Manuscript Draft--
Manuscript Number: IJBIOMAC-D-23-13059R1
Article Type: Research Paper
Section/Category: Carbohydrates, Natural Polyacids and Lignins
Keywords: β-carotene bioaccessibility; Pectic polymers; homogalacturonan
Abstract: The impact of pectin structure on carotenoid bioaccessibility is still uncertain. This
study aims to investigate how the different pectic polymers affected the bioaccessibility
of carotenoids in a simulated juice model during static in vitro digestion. This study
includes homogalacturonan (HG), which is a linear pectic polymer,
rhamnogalacturonan-I (RG-I), which is a branched pectic polymer, and
rhamnogalacturonan (RG), which is a diverse pectic polymer rich in RG-I,
rhamnogalacturonan-II (RG-II), and xylogalacturonan domains. Juice models without
pectin had the highest carotenoid bioaccessibility, suggesting pectin has negative
effects on carotenoid bioaccessibility. During the intestinal phase, systems with HG
showed the highest viscosity, followed by systems with RG and systems with RG-I.
Systems with RG-I had lower carotenoid bioaccessibility than systems with HG and
RG-II. Both the percentage of RG-I and the average side chain length of RG-I had
negative correlations with carotenoid bioaccessibility. RG-I side chains
with more arabinose and/or galactose might cause lower carotenoid bioaccessibility in
this juice model system.
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Conflict of interest
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
Response to Reviewers
Response to Reviewer Comments
Manuscript Number: IJBIOMAC-D-23-13059
Title: Effects of pectin structure on carotenoid bioaccessibility in simulated juice
model
Journal: International Journal of Biological Macromolecules
Dear Prof. Corsaro,
Thank you for your time and effort in handling our manuscript. We would also like to
express our gratitude to the reviewers for their insightful comments on our manuscript.
Their feedback has been invaluable in improving the quality of our work. In response
to their comments, we have made revisions to the manuscript, and the changes have
been highlighted in red. Point-by-point responses to the reviewers’ comments are
listed below this letter.
We believe that the revisions have greatly strengthened the manuscript, and we are
confident that the revised version meets the journal's standards and will be
well-received by the readers. Thank you once again and we look forward to hearing
from you.
Best regards,
Jinfeng Bi
The responses to the reviewers’ comments are as follows:
Reviewer 1:
Comment 1.
Highlights: in the final highlight is it possible to indicate how RG-I affects
bioavailability? Positively/ negatively?
Response:
Thanks for your kind comments. The side chain length of rhamnogalacturonan-I
negatively affects carotenoid bioaccessibility. Due to the word limitation of each
bullet point, it was rewritten as: Longer side chains of rhamnogalacturonan-I reduce
carotenoid bioaccessibility.
Comment 2.
Abstract - can you be more clear on what your mean by RG? It is it not clear whether
this is the same as RG-II or different, try to be more clear in the nomenclature. Could
you suggest - how RG-I causes lower bioavailability in the abstract?
Response:
The RG was clarified in the Abstract (Line 28-29). This study includes
homogalacturonan (HG), which is a linear pectic polymer, rhamnogalacturonan-I
(RG-I), which is a branched pectic polymer, and rhamnogalacturonan (RG), which is
a diverse pectic polymer rich in RG-I, rhamnogalacturonan-II (RG-II), and
xylogalacturonan domains.
We have suggested how RG-I causes lower bioavailability in the Abstract (Line
35-37). RG-I side chains with more arabinose and/or galactose might cause lower
carotenoid bioaccessibility in this juice model system.
Comment 3.
Line 50 - what is the proportion of XGA?
Response:
The proportion of XGA has been added in the Introduction (Line 51-52).
Line 51-52: XGA is an HG substituted at O-3 with a β-linked xylose, and it is found
in pectin in relatively small proportions (<7%).
Comment 4.
Line 75 - could you add a reference?
Response:
References have been added in Line 78.
Comment 5.
Lines 88 - 89 - the pectins are from different sources, could this influence your
findings?
Response:
We acknowledge that pectin structure can vary depending on the sources. The aim of
our study is to investigate the different pectin subdomains on carotenoid
bioaccessibility. Different sources contribute to the different pectin subdomains,

which makes it possible for further exploration of their effects on carotenoid
bioaccessibility. If pectins are from the same sources, this may be challenging to
provide distinct pectin subdomains.
Comment 6.
Line 90 - do you know DE or DM?
Response:
This polygalacturonic acid product contains approximately 5% methylation according
to the description from Megazyme. We have added this information in the Materials
(Line 90).
Comment 7.
Line 102 - mixer rather than machine
Response:
“Machine” has been modified into “mixer” (Line 103).
Comment 8.
Line 109 - could you add a table with the sample information or add this to Table 1?
Response:
We have added a table with sample information (Table 1).
Comment 9.
Line 145 - 146 - number average molar mass (Mn).
Response:
This sentence has been modified according to your comment (Line 147).
Comment 10.
Line 161 - did you measure the zeta-potential of the pectin solutions?
Response:
Yes, we measured the zeta-potential of the pectin solutions with β-carotene and these
values are presented in Fig. 3A as initial zeta-potential values. We have added this in
the Materials & Methods (Line 163-164).
Comment 11.
Line 202 - can you estimate DE from the IR spectra? Are the pectins methylated,
acetylated or both?
Response:
Yes, we can estimate the degree of methyl-esterification (DM) from IR spectra, and
we have added this estimated DM values in Line 205-208.
Line 205-208: The ratio of peak area at 1745 cm-1 (related to methyl ester carbonyl
group stretching) to the sum of the peak areas at 1745 cm-1 and 1608 cm-1 (related to
carboxylate group stretching) was calculated as the degree of methyl-esterification
(DM) [24,25]. The calculated DM of HG and RG-I were 8% and 4%, respectively.
Comment 12.
Lines 188 - 189 - some repetition
Response:
We have modified the sentence according to your comment (Line 190-191).
Line 190-191: The experiments were conducted in triplicate, and each sample was
analyzed three times to ensure accuracy and reproducibility.
Comment 13.
Line 205 - is it that simple? Do different functional groups have the same sensitivity?
Could you add a reference?
Response:
Yes, we have added references in Line 205-208, and this method has been verified as
an effective means to estimate the degree of methyl-esterification (DM) of pectin.
Line 205-208: The ratio of peak area at 1745 cm-1 (related to ester carbonyl group
stretching) to the sum of the peak areas at 1745 cm-1 and 1608 cm-1 (related to
carboxylate group stretching) was calculated as DM [24,25].
[24] G.D. Manrique, F.M. Lajolo, FT-IR spectroscopy as a tool for measuring degree of methyl
esterification in pectins isolated from ripening papaya fruit. Postharvest Biol. Technol. 25(1) (2002)
99-107.
[25] C. Kyomugasho, S. Christiaens, A. Shpigelman, A.M. Van Loey, M.E. Hendrickx, FT-IR
spectroscopy, a reliable method for routine analysis of the degree of methylesterification of pectin in
different fruit-and vegetable-based matrices. Food Chem. 176 (2015) 82-90.

Comment 14.
Figure 2 - Could you redraw this using the conventional symbols for the
monosaccharides?
Response:
Please find the updated Fig. 2 with conventional symbols for the monosaccharides.
Comment 15.
Line 242 - should it be rhamnification?
Response:
Yes, it is. We have now corrected the word (Line 245).
Comment 16.
Line 243 - this is the third figure you have mentioned so it should be Figure 3.
Response:
We have modified this into Fig. 3 (Line 246).
Comment 17.
Line 245 - do you have any information about Rg and hence conformation from your
MALS data? This might help to explain some of your viscosity data.
Line 280 - this depends on conformation see my comment for line 245.
Response:
It’s a pity that we don’t have the data regarding Rg. In our forthcoming study, we will
conduct Rg measurements and establish a correlation with viscosity values.
Comment 18.
Line 255 - check your wording
Response:
We have modified this sentence (Line 258-259).
Line 258-259: Different physical indicators were estimated to investigate the effects
of pectic polymers on digestion process.
Comment 19.
Line 259 - what about the GalA content? This will impart negative charge.
Response:
We have added more discussion regarding GalA content (Line 264-266).
Line 264-266: Typically, an increased GalA content in pectin results in a higher
negative charge [33]. Additionally, more demethylesterified pectin can carry more
negative charges [34].
Comment 20.
Line 269 - "...pectin is close to zero.."
Response:
This sentence has been modified (Line 274).

Comment 21.
Figure 4 - could you redraw this as Figures 4A and 4B as it is not very clear as it is
drawn.
Response:
Yes, it is more clear to present the data in two separated figures. We have redrawn this
figure as Fig. 5A and Fig. 5B according to your comment.
Comment 22.
Line 320 - "... bioavailability compared to other systems..."
Response:
This sentence has been modified (Line 325).
Comment 23.
Line 321 - it is not clear whether this is good or bad, could you be more explicit.
Response:
We have added more explanation and make it more explicit (Line 325-327).
Line 325-327: SS had the highest β-carotene bioaccessibility compared to other
systems (SS-HG, SS-RG-I and SS-RG), which signified that the presence of pectin
could reduce the bioaccessibility carotenoids, thus limiting their potential health
effects.
Comment 24.
Line 342 - did you measure ferulic acid content or protein content?
Response:
We did not measure the ferulic acid content or protein content in this study. Therefore,
we have removed this statement from the manuscript.
Comment 25.
Line 354 - what are the units/ %?
Response:
We have added the unit (%) in Line 357.
Comment 26.
Lines 357 - 358 - "...helpful in increasing ..."
Line 370 - "... SS-RG-I had lower ..."
Response:
These sentences have been modified (Line 361 and 374).
Comment 27.
Line 373 - you might need to be careful with unique as the RG-I structure is an
average of a very broad range of polymers which are very different. For example, PDI
= 2.11, but this is only the heterogeneity in terms of molar mass, there are
heterogeneities in terms of composition and perhaps even conformation. Therefore, it
is perhaps not appropriate to call the structure "unique".

Response:
Yes, you are correct. We have removed the term "unique" from the entire manuscript.
Reviewer 2:
In this manuscript, three pectin-rich polysaccharides which were rich in HG, RG-I and
RG were used to analyze how the different pectic polymers affected the
bioaccessibility of carotenoids in a simulated juice model during static in vitro
digestion. Some issues were listed below.
Comment 1.
To detect how different pectic polymers affected the bioaccessibility of carotenoids, it
is suggested to purify and prepare pectin domains which are composed of one major
type of pectin, e.g. HG, RG-I, RG-II and XGA. In this study, the RG-I and RG
samples are both complex, which were not suitable for the study purpose.
Response:
Thanks for your kind comments. Complex pectin samples, which closely mimic the
natural composition of fruits and vegetables, can offer valuable insights into the
interactions and effects of multiple pectic polymers on carotenoid bioaccessibility.
Furthermore, the technical challenges and limited availability of pure RG-I and RG
samples make them less suitable for this study. As you suggested, the purification and
preparation of pure pectin subdomains might be our further study.
Comment 2.
If RG-II domain also existed in RG-I (potato)? Based on monosaccharide composition,
this pectin seemed to contain more HG than RG-I.
Response:
According to the monosaccharide composition (70.9% GalA, 19.8% Gal, 6.3%Rha,
1.8% Ara, 1.05% Xyl and 0.14% Fuc), RG-II domain seems not existed or existed in a
very small amount in RG-I rich pectin polymers.
The structural framework of RG-I is composed of repeating disaccharides of GalA
and rhamnose. By analyzing the sugar ratios, it has been determined that the RG-I rich
pectin polymer constitutes 34% of RG-I, whereas the HG rich pectin polymer
contains only 7% RG-I. Therefore, in our study, the RG-I derived from potatoes can
be classified as a pectin polymer with a relatively higher abundance of RG-I.
Comment 3.
How to prove RG-II domain were present in RG (soybean)?
Response:
As fucose exist in the pectin polymers, so it is assumed the RG-II domain is present in
RG (soybean).
Comment 4.
In line 228, how to determine the ratios of different types of pectin?
Response:
The composition ratios were calculated based on the mol% quantifiable of GalA and
neutral sugars as follows:
2×Rha+Ara+Gal
The percentage of RG-I (% RG-I) = GalA+Rha+Ara+Gal+Xyl+Fuc
The percentage of XGA is calculated as the mol% of Xyl, and the percentage of RG-II
is estimated as the mol% of the remaining monosaccharides.

Abstract
Abstract
The impact of pectin structure on carotenoid bioaccessibility is still uncertain. This
study aims to investigate how the different pectic polymers affected the
bioaccessibility of carotenoids in a simulated juice model during static in vitro
digestion. This study includes homogalacturonan (HG), which is a linear pectic
polymer, rhamnogalacturonan-I (RG-I), which is a branched pectic polymer, and
rhamnogalacturonan (RG), which is a diverse pectic polymer rich in RG-I,
rhamnogalacturonan-II (RG-II), and xylogalacturonan domains. Juice models without
pectin had the highest carotenoid bioaccessibility, suggesting pectin has negative
effects on carotenoid bioaccessibility. During the intestinal phase, systems with HG
showed the highest viscosity, followed by systems with RG and systems with RG-I.
Systems with RG-I had lower carotenoid bioaccessibility than systems with HG and
RG-II. Both the percentage of RG-I and the average side chain length of RG-I had
negative correlations with carotenoid bioaccessibility. RG-I side chains with more
arabinose and/or galactose might cause lower carotenoid bioaccessibility in this juice
model system.
Keywords: β-carotene bioaccessibility; Pectic polymers; Homogalacturonan;

Rhamnogalacturonan-I; Rhamnogalacturonan-II; Digestive fluids
Revised manuscript (clean version) Click here to view linked References
1 Effects of pectin structure on carotenoid bioaccessibility in

2 simulated juice model
3
4 Jianing Liua,b, Jinfeng Bia,*, Xuan Liua,*, Dazhi Liua,c, Vincenzo Foglianob, Matthijs
5 Dekkerb, Ruud Verkerkb
a
7 Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences
8 (CAAS), Key Laboratory of Agro-Products Processing, Ministry of Agriculture and
9 Rural Affairs, Beijing 100193, China
b
10 Food Quality and Design Group, Wageningen University & Research, Bornse
11 Weilanden 9, 6708 WG, Wageningen, the Netherlands
c
12 Laboratory of Food Chemistry, Wageningen University & Research, Bornse
14
15
16
*
17 Corresponding author.
18 Tel.:+86-1062812584.
19 E-mail address: bijinfeng2010@163.com and liuxuancaas@126.com.
20 ADD: No. 2, Yuanmingyuan West Road, Haidian District, Beijing, 100193, China.
21
1
22 Abstract
23 The impact of pectin structure on carotenoid bioaccessibility is still uncertain. This
24 study aims to investigate how the different pectic polymers affected the bioaccessibility
25 of carotenoids in a simulated juice model during static in vitro digestion. This study
26 includes homogalacturonan (HG), which is a linear pectic polymer,
27 rhamnogalacturonan-I (RG-I), which is a branched pectic polymer, and
28 rhamnogalacturonan (RG), which is a diverse pectic polymer rich in RG-I,
29 rhamnogalacturonan-II (RG-II), and xylogalacturonan domains. Juice models without
30 pectin had the highest carotenoid bioaccessibility, suggesting pectin has negative
31 effects on carotenoid bioaccessibility. During the intestinal phase, systems with HG
32 showed the highest viscosity, followed by systems with RG and systems with RG-I.
33 Systems with RG-I had lower carotenoid bioaccessibility than systems with HG and
34 RG-II. Both the percentage of RG-I and the average side chain length of RG-I had
35 negative correlations with carotenoid bioaccessibility. RG-I side chains with more
36 arabinose and/or galactose might cause lower carotenoid bioaccessibility in this juice
37 model system.
38 Keywords: β-carotene bioaccessibility; Pectic polymers; Homogalacturonan;
39 Rhamnogalacturonan-I; Rhamnogalacturonan-II; Digestive fluids
2
40 Introduction
41 Pectin is a polysaccharide found in plant cell walls, predominantly consisting of
42 1,4-linked-α-D-galacturonic acid units. The primary structural domains of pectin are
43 homogalacturonan (HG), rhamnogalacturonan-I (RG-I), rhamnogalacturonan-II (RG-
44 II), and xylogalacturonan (XGA). HG makes up approximately 65% of the pectin
45 molecule, and it contains a linear chain of galacturonic acid (GalA) with methyl-
46 esterified carboxyl groups. RG-I is the second most abundant pectic polymer (i.e. 20-
47 35% of pectins), which comprises a backbone of repeating GalA and rhamnose
48 disaccharides with arabinan, galactan, and/or arabinogalactan side chains. The most
49 structurally complex pectin, RG-II, makes up about 10% of pectin. The backbone of
50 RG-II is made up of HG, with intricate side chains including simple sugars that are
51 connected to the GalA. XGA is an HG substituted at O-3 with a β-linked xylose, and it
52 is found in pectin in relatively small proportions (<7%) [1,2].
53 Different structural characteristics of pectic polymers contribute to their different
54 functional properties and bioactivities [3,4]. Many of pectin's functional characteristics
55 are influenced by the quantity of esterified GalA residues and their distribution in HG.
56 When methyl-ester groups are removed from HG, it becomes calcium cross-linkable,
57 allowing the formation of supramolecular assemblies and gels [5]. Furthermore, earlier
58 research has shown that cooperative cation binding to pectin requires at least 6 to 10
59 consecutive non-methyl-esterified GalA units [6,7]. RG-I pectin is heterogeneous due
60 to its variable glycosyl linkages, monosaccharide composition of side chains, chain
61 lengths, and distribution. Ferulic acid groups alter the molecular weight in the cell wall
3
62 and lead to poor pectin gelation properties by promoting the cross-linking of neutral
63 sugars in RG-I through ferulic bridges [8]. Recently, the research focused on the
64 importance of the pectin hairy region. Pectin RG-I portion showed improved
65 bioactivities, including inhibition of cell migration, and immunological and prebiotic
66 activities [9,10]. In vitro tests with human colon adenocarcinoma (Caco-2) cells showed
67 that RG-II had promisingly strong anti-cancer proliferation activities [11].
68 During the upper gastrointestinal stage, pectin may affect the digestion and
69 absorption of lipophilic carotenoids, by inhibiting lipid digestion [12]. Compared with
70 high pectin concentration, low pectin concentration favored the binding of bile salt and
71 carotenoid micellization [13]. The degree of lipid digestion decreased with an
72 increasing degree of pectin methyl-esterification [14]. Several studies have reported
73 carotenoid bioaccessibility and pectin structural and functional characteristics
74 (galacturonic acid concentration, degree of methyl-esterification and acetylation, and
75 emulsifying activity) of juices are related [15,16], however, the molecular mechanism
76 underpinning this relationship is still uncertain. It is possible that specific pectic
77 polymers are actually responsible for the reduction of carotenoid bioaccessibility
78 [12,16]. Therefore, the aim of this study was to investigate the influences of different
79 pectic polymers on carotenoid bioaccessibility of simulated juice models using static in
80 vitro digestion. In the present study, the pectic polymers include HG domain-rich pectic
81 polymer, RG-I region-rich pectic polymer, and rhamnogalacturonan (RG) pectic
82 polymer, which is more diverse with RG-I, RG-II and XGA domains. The simulated
83 juice models were applied to directly understand how pectin polymers influences β-
4
84 carotene digestion. Viscosity and ζ-potential of digestive fluids, β-carotene retention
85 and β-carotene bioaccessibility were recorded, to gain better knowledge of the digestive
86 process.
87
88 1. Materials and methods
89 2.1 Materials
90 Polygalacturonic acid (citrus pectin, ~5% methylation), rhamnogalacturonan I
91 (potato), and rhamnogalacturonan (soybean) were purchased from Megazyme (Bray,
92 Ireland). Polygalacturonic acid is seen as an HG domain-rich pectic polymer.
93 Rhamnogalacturonan I is considered to be an RG-I region-rich pectic polymer.
94 Rhamnogalacturonan is considered a more diverse pectic polymer rich in RG-I, RG-II
95 and XGA domains. Porcine gastric mucin, pepsin from porcine gastric mucosa, lipase
96 from porcine pancreas, β-carotene (≥ 93%), β-carotene (HPLC grade) and butylated
97 hydroxytoluene (BHT) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
98
99 2.2 Methods
100 2.2.1 Juice model system preparation
101 The juice model system was prepared according to our previous study, with minor
102 modifications [17]. β-carotene (30 mg) in 100 mL acetic acid-sodium acetate buffer (0.2
103 M, pH=6) with 0.2% corn oil (w/w) was dispersed using a high-speed dispersion mixer
104 (T25 Ultra-TURRAX, IKA, Germany) at 14,000 rpm for 40 min. Subsequently, HG
105 (polygalacturonic acid), RG-I (rhamnogalacturonan I), and RG (rhamnogalacturonan),
5
106 at a proportion of 1% (w/v), were added to the β-carotene solution, and fully mixed
107 using the Ultra-TURRAX at 7,000 rpm for 10 min. The systems were then adjusted to
108 pH 6 and homogenized at 250 MPa (SPCH-EP-IC-16-30, Homogenising Systems Ltd,
109 London, UK). Simulated systems containing β-carotene, β-carotene with HG, RG-I, or
110 RG were abbreviated as SS, SS-HG, SS-RG-I, and SS-RG, respectively (Table 1).
111
112 2.2.2 Structural characterization of pectic polymers
113 2.2.2.1 Fourier transform infrared spectroscopy (FT-IR) analysis
114 The FT-IR spectra of pectic polymers were determined using an FT-IR
115 spectrometer (Tensor 27, Bruker, Germany) in the wavenumber region 4000~400 cm-1
116 at a resolution of 4 cm-1 and a cumulative scan of 64. Before FTIR analysis, pectic
117 polymers were pulverized and pressed into flake with KBr at a ratio of 1:100 (w/w).
118
119 2.2.2.2 Determination of galacturonic acid (GalA) and neutral sugar concentration
120 GalA and neutral sugar concentrations were performed according to the previous
121 study [16]. High-performance anion-exchange chromatography (HPAEC) was achieved
122 using ICS-3000 system (Dionex, Sunnyvale, CA, USA) equipped with a CarboPacTM
123 PA20 column and a pulsed amperometric detector. Monosaccharides including GalA,
124 galactose (Gal), arabinose (Ara), rhamnose (Rha), xylose (Xyl), and fucose (Fuc) were
125 used for identification and quantification.
126 To obtain information on the domains of the pectic polymers, five composition
127 ratios were calculated based on the mol% quantifiable of GalA and neutral sugars, with
6
128 some modifications [18,19]:
GalA+Rha
129 The linearity of pectic polymers = (1)
GalA+Rha+Ara+Gal+Xyl+Fuc
GalA−Rha
130 The percentage of HG (% HG) = (2)
GalA−Rha
131 The ratio of HG to RG-I backbone = (3)
2×Rha
2×Rha+Ara+Gal
132 The percentage of RG-I (% RG-I) = (4)
Ara+Gal
133 The average side chain length of RG-I = (5)
Rha
134
135 2.2.2.3 Determination of molar mass distribution
136 The molar mass distributions of pectins were determined using high-performance
137 size exclusion chromatography (HPSEC) coupled with multiangle laser light scattering
138 (MALLS) (Dawn Heleos II, Wyatt Technology, Santa Barbara, CA, USA), reflective
139 index (RI) detector (Optilab rEX, Wyatt Technology, Santa Barbara, CA, USA) and
140 ultraviolet (UV) detector (L-2400, Hitachi, Tokyo, Japan). The dn/dc value (refractive
141 index increment) was 0.146 mL/g. Pectins (10 mg) were dissolved in eluent (0.1 M
142 NaCl) overnight and filtrated through a 0.45 μm filter membrane before analysis.
143 Samples (100 μL) were injected into an OHpak SB-806M HQ column (8.0 × 300 mm,
144 Shodex, Tokyo, Japan). Elution was performed with 0.1 M NaCl solution at a flow rate
145 of 0.5 mL/min for 30 min and the column was kept at 35 °C. The polydispersity index
146 (PDI) was calculated as the ratio of the weight-average molar mass (Mw) to the number
147 average molar mass (Mn).
148
149 2.2.3 Static in vitro digestion
7
150 Juice model systems were subjected to an in vitro digestion that encompassed
151 simulated oral, gastric, and intestinal phases [20]. During intestine digestion stage, an
152 automatic titration facility (Metrohm, USA, Inc.) was utilized to maintain pH at 7.0 by
153 adding 0.25 M NaOH solution over 2 h at 37°C. To stop the enzymatic reaction, samples
154 were placed on ice after each digestion phase.
155
156 2.2.4 Digestive fluid characteristics
157 2.2.4.1 Viscosity property of the digestive fluids
158 The viscosity of the digestive fluids at a shear rate of 50 s-1 was measured by using
159 a Physica MCR 301 rheometer (Anton Paar, Graz, Austria). The parallel plate geometry
160 had a 40 mm diameter and the gap size was 1 mm.
161
162 2.2.4.2 ζ-potential of the digestive fluids
163 The ζ-potential values of the initial systems (pectin solutions with β-carotene) and
164 their digestive fluids during digestion were measured by using a Zetasizer Nano ZS
165 analyzer (Malvern Instruments Ltd., Malvern, UK).
166
167 2.2.5 β-carotene bioaccessibility and retention ratio
168 After the above digestion process, the raw intestinal digesta was centrifuged at
169 8000 rpm (8801×g) and 4 °C for 1 h. β-carotene bioaccessibility was calculated as
170 follows:
171 β-carotene bioaccessibility (%)=(Cmicelle/Craw digesta) ×100 (6)
8
172 where Cmicelle and Craw digesta are the contents of β-carotene in the micelle fraction and
173 the raw digesta, respectively.
174 The β-carotene retention ratio during each digestion phase was calculated as
175 follows:
176 β-carotene retention ratio (%)=Cdigestion/Cinitial×100 (7)
177 where Cdigestion and Cinitial are the β-carotene concentrations (μg/mL) in different systems
178 during each digestion phase and in the initial system, respectively.
179
180 2.2.6 Extraction and determination of β-carotene
181 A previous procedure was used to extract and quantify β-carotene [21]. Samples
182 were separated and analyzed on high-performance liquid chromatography (HPLC)
183 system with a reversed-phase C30-column (250 mm × 4.6 mm, 5 μm; YMC Europe,
184 Dinslaken, Germany), a binary HPLC pump (1525, Waters, Milford, MA, USA) and a
185 photodiode array detector (2998, Waters, Milford, MA, USA). The gradient program,
186 detection wavelength, flow rate of mobile phase were performed according to a
187 previous procedure [16].
188
189 2.2.7 Statistics
190 The experiments were conducted in triplicate, and each sample was analyzed three
191 times to ensure accuracy and reproducibility. All data were presented as mean ±
192 standard deviation. Analysis of variance (ANOVA) followed by a Tukey test was used
193 to determine significant differences using R software (Version 4.2.0). The Pearson
9
194 correlation analysis was carried out by the “corrplot” package in R software.
195
196 2. Results and discussion
197 2.1 Structural characterization of pectin polymers
198 3.1.1 Functional groups of pectic polymers
199 As shown in Fig. 1A, the following were the main characteristics of the absorption
200 peaks: (i) The absorption peak at ≈ 3364 cm-1 was attributed to O-H stretching vibration,
201 and the peak at ≈ 2936 cm-1 was caused by C-H stretching of CH2 groups; (ii) the peaks
202 at ≈ 1745 cm-1 and 1608 cm-1 corresponded to the C=O stretching vibration of methyl-
203 esterified carboxyl groups and C=O asymmetrical stretching vibration of free carboxyl,
204 respectively; (iii) the peak at ≈ 1418 cm-1 was ascribed to a symmetric carboxylate
205 stretch [22,23]. The ratio of peak area at 1745 cm-1 (related to methyl ester carbonyl
206 group stretching) to the sum of the peak areas at 1745 cm-1 and 1608 cm-1 (related to
207 carboxylate group stretching) was calculated as the degree of methyl-esterification (DM)
208 [24,25]. The calculated DM of HG and RG-I were 8% and 4%, respectively. However,
209 the absorption area of RG at 1745 cm-1 was not detected, which suggested that methyl-
210 esters were not present in the RG chain. The variation of the DM observed could be
211 attributed to the different extraction methods applied.
212
213 3.1.2 Monosaccharide content and composition ratios of pectic polymers
214 As shown in Fig. 1B, the GalA content of HG was the highest (94.1%), followed
215 by RG-I (70.9%) and RG (50.9%). HG is composed of 94% GalA and 6% other
10
216 monosaccharides, such as 3.9% Gal and 1.5% Rha. RG-I comprises 70.9% GalA,
217 followed by 19.8% Gal, 6.3% Rha, and 1.8% Ara that constitute the RG-I region. RG
218 is a more diverse pectic polymer compared to HG and RG-I, as evidenced by its
219 composition of 50.9% GalA, 16.9% Xyl, and 13.1% Fuc. Individual monosaccharide
220 contents are not sufficient to gain insight into the pectin chains. With the help of the
221 ratios between the composing monosaccharides, the overall structure of pectin chains
222 becomes more visible.
223 As shown in Table 2, compared to the other two pectic polymers, HG is composed
224 of more linear/less branched pectin evidenced by the higher linearity of pectic
225 polymers, % HG and the ratio of HG to RG-I backbone. The higher linearity of HG
226 makes it potential to have higher Ca2+-binding capability, due to the higher possibility
227 of consecutive non-methyl-esterified GalA residues. RG-I is composed of a more
228 branched RG-I domain according to higher % RG-I and average side chain length of
229 RG-I. Pectin polymers rich in RG-I side chains have better emulsion stability by
230 forming a hydrated layer [26]. RG is a pectic polymer with more diverse domains,
231 which consists of 26.87% RG-I and approximately 13.14% RG-II side chains and 16.94%
232 XGA side chains. The percentage of the RG-II and XGA backbones is unclear, though,
233 as both of their backbones are HG. Based on the obtained structural characteristics, the
234 structures of the pectic polymers are visualized as hypothetical model structures
235 presented in Fig. 2. These distinct monosaccharide compositions among different pectic
236 polymers make it possible for further investigation of the effects of pectic polymers on
237 carotenoid bioaccessibility.
11
238
239 3.1.3 Molar mass distribution of pectic polymers
240 Mw and PDI values are presented in Table 2, and a higher PDI represents a wider
241 molecular mass distribution. RG showed the highest Mw, followed by RG-I and HG.
242 HG showed the lowest PDI values compared to RG and RG-I. The rich side chains of
243 RG-I and RG might contribute to their higher Mw and wider molecular mass distribution.
244 According to correlation analysis, the linearity of pectin had a negative correlation with
245 Mw, indicating that a higher degree of rhamnification might correspond to a larger Mw
246 of the polysaccharide (Fig. 3). Many pectin properties are linked to its Mw, and high
247 molecular mass polymers generally contribute to promising functionalities, such as
248 emulsifying and emulsion stabilizing abilities [27]. In most cases, higher Mw
249 macromolecules obstruct fluid motion, resulting in higher viscosity solutions [28]. It
250 should be noted that other factors, such as pectin sources and pectin fractions, might
251 also affect the viscosity of pectins [29,30]. Decreasing Mw might reduce the maximum
252 amount of Ca2+ that pectin can bind [31], however, the calcium binding ability also
253 depends on the degree and distribution pattern of methyl-ester groups among HG region
254 [32].
255
256 3.2 Physical characteristics of digestive fluids
257 3.2.1 ζ-potential of digestive fluids
258 Different physical indicators were estimated to investigate the effects of pectic
259 polymers on digestion process. Generally, changes in the ζ-potential of particles can
12
260 influence their electrostatic repulsion or attraction, which in turn can affect the stability
261 and behavior of the systems during digestion. As seen in Fig. 4A, initial ζ-potential
262 values of SS-HG and SS-RG were lower than those of SS and SS-RG-I, which
263 suggested that SS-HG and SS-RG were enveloped by more negatively charged
264 substances. Typically, an increased GalA content in pectin results in a higher negative
265 charge [33]. Additionally, more demethylesterified pectin can carry more negative
266 charges [34]. During the oral phase, the ζ-potential value of SS was the highest,
267 followed by SS-RG, SS-RG-I and SS-HG. The ζ-potential values increased from the
268 initial phase to the gastric phase, while decreased in the intestinal phase. The highly
269 acidic conditions, which cause the protein-coated droplets to be positive, and the
270 presence of anionic mucin, which binds to the cationic protein surfaces, are both related
271 to the increase in ζ-potential during the gastric phase [35,36]. Specifically, GalA, the
272 degree of methyl-esterification and the distribution of methyl esters of pectin influence
273 their interactions with protein [37]. Moreover, the pectin surface charge decreased with
274 the decreasing pH, and at pH 2, the ζ-potential of pectin is close to zero [38]. The
275 amount of the negative charge on the backbone of pectin decreased as a result of
276 protonation (a drop in pH). The fact that the ζ-potential values changed from their initial
277 positive values to more negative ones during the intestinal phase could be attributed to
278 a number of negatively charged species, including free fatty acids released during
279 lipolysis, intricate colloidal structures, undigested oil, and anionic polymers [14].
280
281 3.2.2 Apparent viscosity of digestive fluids
13
282 The apparent viscosity of digestive fluids is given in Fig. 4B. The physical feature
283 of apparent viscosity is linked to soluble dietary fibers, particularly pectic
284 polysaccharides. Initial SS-HG and SS-RG showed higher viscosity than SS-RG-I.
285 Generally, pectin with a higher Mw has a higher viscosity [28]. Moreover, HG pectin
286 contributed greatly to the viscosity of pectin [39]. There was a significant negative
287 correlation between the initial viscosity of the systems and the RG-I percentage (Fig.
288 3). Due to the neutral sugar side chains, solutions containing high contents of RG-I
289 and/or RG-II pectin usually have lower viscosity values than solutions with high GalA
290 content [39]. From the initial phase to the gastric phase, the viscosity of the four systems
291 all decreased, which might be caused by the dilution effect of digestive fluids. These
292 results are consistent with those obtained with broccoli-digested fractions, where
293 viscosity decreased from the gastric phase to the intestinal phase [40]. During the
294 intestinal phase, SS-HG showed the highest viscosity, followed by SS-RG, SS-RG-I
295 and SS. A negative correlation has been found between the HG to RG-I ratio and the
296 viscosity of the systems during the intestinal phase. The interactions between pectic
297 polymers and the digestive fluids might also contribute to the results, especially the
298 crosslinking between pectin and digestive enzymes.
299
300 3.3 β-carotene retention ratio during digestion phases
301 As seen in Fig. 5, in the oral phase, the β-carotene retention ratio of SS-RG was
302 lower than that of SS, while in the gastric phase, the β-carotene retention ratio of SS-
303 RG was the highest compared with other systems. This suggested that SS-RG might be
14
304 more stable under acidic conditions. At the intestinal phase, the β-carotene retention
305 ratios of SS-HG, SS-RG-I, and SS-RG had no significant difference. Carotenoids are
306 isoprenoid pigments with a polyene backbone that contains a variable number of
307 conjugated double bonds, therefore, they are prone to be oxidized. A previous study
308 showed that the stability of carotenoids at different processing can be significantly
309 improved in an oil-based system, compared with a water-based juice system [41]. In
310 our study, the carotenoids were in a homogenized juice model system, which is similar
311 to a water-based juice system, therefore, carotenoid stability might be affected by high-
312 pressure homogenization to some extent. A negative correlation was observed between
313 the β-carotene retention ratio during the intestinal phase and the ζ-potential values of
314 initial systems. This suggests that a higher negative charge could potentially enhance
315 the delivery of carotenoids during the intestinal stage. Additionally, a negative
316 correlation was found between the β-carotene retention ratio and the viscosity of
317 digestive fluids during the intestinal phase. Therefore, the pectin-induced viscosity
318 might have effects on the β-carotene stability during the intestinal stage.
319
320 3.4 β-carotene bioaccessibility
321 Carotenoids need to be released from the food matrix, solubilized into the lipid
322 phase, and incorporated into mixed micelles in order to be absorbed via the human
323 intestinal epithelium [12]. The levels of carotenoids available for intestinal absorption,
324 specifically the carotenoids in mixed micelles, are referred to as bioaccessible
15
325 carotenoids. SS had the highest β-carotene bioaccessibility compared to other systems
326 (SS-HG, SS-RG-I and SS-RG), which signified that the presence of pectin could reduce
327 the bioaccessibility carotenoids, thus limiting their potential health effects. Similar
328 observations were also found by Cervantes-Paz et.al [13], in which high pectin
329 concentration decreased carotenoid micellization. Pectin can increase the viscosity of
330 digestive fluids, allowing digesta to last longer in the gastrointestinal phase while
331 simultaneously reducing the movement of substrates and digestive enzymes (eg., pepsin
332 and lipase), decreasing lipase activity and micelle generation [42]. Moreover, different
333 pectic polymers caused different influences on carotenoid bioaccessibility. SS-HG and
334 SS-RG had similar β-carotene bioaccessibility, while SS-RG-I had a smaller β-carotene
335 bioaccessibility than those of SS-HG and SS-RG. Furthermore, both % RG-I and the
336 average side chain length of RG-I had negative correlations with carotenoid
337 bioaccessibility. Therefore, it was deduced that more RG-I side chains with Ara and/or
338 Gal might cause lower carotenoid bioaccessibility in this juice model system. These
339 results are consistent with those obtained with carrot juice, where higher emulsifying
340 properties caused a lower value of total carotenoid bioaccessibility [16]. The proposed
341 mechanisms of the effects of pectic polymers on carotenoid bioaccessibility are shown
342 in Fig. 6. The lipid hydrolysis produces monoacylglycerols and free fatty acids, which
343 together with bile salts contribute to the formation of mixed micelles. In this context,
344 lipase can greatly increase lipolysis and carotenoid bioaccessibility [43]. Pectin
345 segments with enriched RG-I/neutral sugar side chains contribute to better emulsifying
346 properties [44,45]. Pectin with better emulsification properties might be able to form
16
347 thicker hydrated layers at the oil-water interface and inhibit lipase to the surface,
348 resulting in low carotenoid bioaccessibility. A recent study based on molecular
349 dynamics simulations also revealed that RG-I and RG-II domains were adsorbed on the
350 oil-water interface in a folded form, creating a thick viscoelastic film while the HG
351 domain was tiled on the interface [46].
352 It should also be mentioned that in real fruit and vegetable systems, RG-I side
353 chains could also be cross-linked to xylans, xyloglucans, lignins, and proteins [5]. The
354 above-mentioned cross-linking among pectic polymers can also influence carotenoid
355 bioaccessibility, but this is not considered in the juice model system in this study.
356 Despite the absence of explicit localization obstacles of carotenoids in model systems,
357 β-carotene bioaccessibility was still comparatively low, ranging from 7.97% to 14.55%.
358 It implies that lipids are important in enhancing β-carotene bioaccessibility since dietary
359 lipids could stimulate bile salt secretion and increase the number of carotenoids carried
360 in micelles. Furthermore, other consumption forms of food products might be helpful
361 in increasing carotenoid bioaccessibility. Making fruit and vegetable-based emulsions
362 has been noted as a promising method to increase carotenoid bioaccessibility by
363 researchers [47,48]. Therefore, further research might focus on the effects of pectic
364 polymers on carotenoid bioaccessibility in model systems with the addition of more
365 lipids.
366
367 4. Conclusions
368 The simulated juice models were helpful to understand the effects of different
17
369 pectic polymers on carotenoid bioaccessibility. The ζ-potential values of all the systems
370 increased from the initial phase to the gastric phase, while decreased in the intestinal
371 phase. When compared to the initial ζ-potential values, ζ-potential became more
372 negative during the intestinal phase. During the intestinal phase, SS-HG showed the
373 highest viscosity, followed by SS-RG, SS-RG-I and SS. SS-HG and SS-RG had similar
374 β-carotene bioaccessibility, while SS-RG-I had a lower β-carotene bioaccessibility than
375 those of SS-HG and SS-RG. Both % RG-I and the average side chain length of RG-I
376 had negative influences on carotenoid bioaccessibility.
377
378 Acknowledgements
379 This work was supported by the National Key Research and Development
380 Program of China (2022YFD1600505), the earmarked fund for China Agriculture
381 Research System (CARS-27), and the Joint PhD Program between Wageningen
382 University & Research (WUR) and Chinese Academy of Agricultural Sciences (CAAS)
383 (No. MOE11NL1A20151701N).
18
384 References
385 [1] D. Mohnen, Pectin structure and biosynthesis, Curr. Opin. Plant Biol. 11(3) (2008)
386 266-277.
387 [2] J. Zandleven, S.O. Sørensen, J. Harholt, G. Beldman, H.A. Schols, H.V. Scheller, &
388 A.J. Voragen, Xylogalacturonan exists in cell walls from various tissues of
389 Arabidopsis thaliana, Phytochemistry. 68(8) (2007) 1219-1226.
390 [3] B. Neckebroeck, S.H.E. Verkempinck, G. Vaes, K. Wouters, J. Magnee, M. E.
391 Hendrickx, et al. Advanced insight into the emulsifying and emulsion stabilizing
392 capacity of carrot pectin subdomains, Food Hydrocolloids, 102 (2020) 105594.
393 [4] P. M. Pieczywek, J. Ciesla, W. Płazinski, A. Zdunek, Aggregation and weak gel
394 formation by pectic polysaccharide homogalacturonan, Carbohydr. Polym. 256
395 (2021) 117566.
396 [5] W.G.T. Willats, L. Mccartney, W. Mackie, & J.P. Knox, Pectin: Cell biology and
397 prospects for functional analysis, Plant Mol. Biol. 47(1-2) (2001) 9-27.
398 [6] P.J.H. Daas, B. Boxma, A.M.C.P. Hopman, A.G.J. Voragen, H.A. Schols,
399 Nonesterified galacturonic acid sequence homology of pectins, Biopolymers. 58(1)
400 (2001) 1-8.
401 [7] G.A. Luzio, R.G. Cameron, Demethylation of a model homogalacturonan with the
402 salt-independent pectin methylesterase from citrus: Part II. structure–function
403 analysis, Carbohydr. Polym. 71(2) (2008) 300-309.
404 [8] A. Oosterveld, I.E. Pol, G. Beldman, A.G. Voragen, Isolation of feruloylated
405 arabinans and rhamnogalacturonans from sugar beet pulp and their gel forming
19
406 ability by oxidative cross-linking, Carbohydr. Polym. 44(1) (2001) 9-17.
407 [9] D. Wu, J. Zheng, G. Mao, W. Hu, X. Ye, R.J. Linhardt, et al. Rethinking the impact
408 of RG-I mainly from fruits and vegetables on dietary health, Crit. Rev. Food Sci.
409 Nutr. 60 (2020) 2938-2960.
410 [10] M.Y. Zhang, J. Cai, Preparation of branched RG-I-rich pectin from red dragon fruit
411 peel and the characterization of its probiotic properties, Carbohydr. Polym. (2023)
412 120144.
413 [11] L. Ai, Y. Chung, S. Lin, K. Lee, P. Lai, Y. Xia, et al. Active pectin fragments of
414 high in vitro antiproliferation activities toward human colon adenocarcinoma cells:
415 Rhamnogalacturonan II, Food Hydrocolloids. 83 (2018) 239-245.
416 [12] B. Cervantes-Paz, J.J. Ornelas-Paz, S. Ruiz-Cruz, C. Rios-Velasco, V. Ibarra-
417 Junquera, E.M. Yahia, et al. Effects of pectin on lipid digestion and possible
418 implications for carotenoid bioavailability during pre-absorptive stages: A review,
419 Food Res. Int. 99 (2017) 917-927.
420 [13] B. Cervantes-Paz, J.D.J. Ornelas-Paz, J.D. Pérez-Martínez, J. Reyes-Hernández,
421 P.B. Zamudio-Flores, C. Rios-Velasco, et al. Effect of pectin concentration and
422 properties on digestive events involved on micellarization of free and esterified
423 carotenoids, Food Hydrocolloids. 60 (2016) 580-588.
424 [14] M. Espinal-Ruiz, L.P. Restrepo-Sanchez, C.E. Narvaez-Cuenca, D.J. McClements,
425 Impact of pectin properties on lipid digestion under simulated gastrointestinal
426 conditions: Comparison of citrus and banana passion fruit (Passiflora tripartita var.
427 mollissima) pectins, Food Hydrocolloids. 52 (2016) 329-342.
20
428 [15] Y. Ding, X. Liu, J. Bi, X. Wu, X. Li, J. Liu, et al. Effects of pectin, sugar and pH
429 on the β-Carotene bioaccessibility in simulated juice systems, LWT--Food Sci.
430 Technol. 124 (2020) 109125.
431 [16] X. Liu, J. Liu, J. Bi, J. Yi, J. Peng, C. Ning, et al. Effects of high pressure
432 homogenization on pectin structural characteristics and carotenoid bioaccessibility
433 of carrot juice, Carbohydr. Polym. 203 (2019) 176-184.
434 [17] J. Liu, J. Bi, X. Liu, D. Liu, X. Wu, J. Lyu, et al. Effects of pectins and sugars on
435 β-carotene bioaccessibility in an in vitro simulated digestion model, J. Food
436 Compos. Anal. 91 (2020) 103537.
437 [18] S.E. Broxterman, P. Picouet, H.A. Schols, Acetylated pectins in raw and heat
438 processed carrots, Carbohydr. Polym. 177 (2017) 58-66.
439 [19] K. Houben, R.P. Jolie, I. Fraeye, A.M. Van Loey, M.E. Hendrickx, Comparative
440 study of the cell wall composition of broccoli, carrot, and tomato: Structural
441 characterization of the extractable pectins and hemicelluloses, Carbohydr. Res.
442 346(9) (2011) 1105-1111.
443 [20] J. Liu, D. Liu, J. Bi, X. Liu, Y. Lyu, R. Verkerk, et al. Micelle separation conditions
444 based on particle size strongly affect carotenoid bioaccessibility assessment from
445 juices after in vitro digestion, Food Res. Int. 151 (2022) 110891.
446 [21] G. Knockaert, L. Lemmens, S. Van Buggenhout, M. Hendrickx, A. Van Loey,
447 Changes in β-carotene bioaccessibility and concentration during processing of
448 carrot puree, Food Chem. 133(1) (2012) 60-67.
449 [22] M. Kazemi, F. Khodaiyan, S.S. Hosseini, Utilization of food processing wastes of
21
450 eggplant as a high potential pectin source and characterization of extracted pectin,
451 Food Chem. 294 (2019) 339-346.
452 [23] J.S. Yang, T.H. Mu, M.M. Ma, Extraction, structure, and emulsifying properties of
453 pectin from potato pulp, Food Chem. 244 (2018) 197-205.
454 [24] G.D. Manrique, F.M. Lajolo, FT-IR spectroscopy as a tool for measuring degree
455 of methyl esterification in pectins isolated from ripening papaya fruit. Postharvest
456 Biol. Technol. 25(1) (2002) 99-107.
457 [25] C. Kyomugasho, S. Christiaens, A. Shpigelman, A.M. Van Loey, M.E. Hendrickx,
458 FT-IR spectroscopy, a reliable method for routine analysis of the degree of
459 methylesterification of pectin in different fruit-and vegetable-based matrices. Food
460 Chem. 176 (2015) 82-90.
461 [26] T. Funami, M. Nakauma, S. Ishihara, R. Tanaka, T. Inoue, G.O. Phillips, Structural
462 modifications of sugar beet pectin and the relationship of structure to
463 functionality, Food Hydrocolloids. 25(2) (2011) 221-229.
464 [27] B. Neckebroeck, S.H.E. Verkempinck, T. Bernaerts, D. Verheyen, M.E. Hendrickx,
465 A. M. Van Loey, Investigating the role of the different molar mass fractions of a
466 pectin rich extract from onion towards its emulsifying and emulsion stabilizing
467 potential, Food Hydrocolloids. 117 (2021) 106735.
468 [28] L. Wan, Q. Chen, M. Huang, F. Liu, S. Pan, Physiochemical, rheological and
469 emulsifying properties of low methoxyl pectin prepared by high hydrostatic
470 pressure-assisted enzymatic, conventional enzymatic, and alkaline de-
471 esterification: A comparison study, Food Hydrocolloids. 93 (2019) 146-155.
22
472 [29] J. Mierczyńska, J. Cybulska, P.M. Pieczywek, A. Zdunek, Effect of storage on
473 rheology of water-soluble, chelate-soluble and diluted alkali-soluble pectin in
474 carrot cell walls, Food Bioprocess Technol. 8 (2015) 171-180.
475 [30] J. Mierczyńska, J. Cybulska, A. Zdunek, Rheological and chemical properties of
476 pectin enriched fractions from different sources extracted with citric
477 acid, Carbohydr. Polym. 156 (2017) 443-451.
478 [31] C. Kyomugasho, C. Munyensanga, M. Celus, K. Dewettinck, A.M. Van Loey, T.
479 Grauwet, M.E. Hendrickx, Molar mass influence on pectin-Ca2+ adsorption
480 capacity, interaction energy and associated functionality: Gel microstructure and
481 stiffness, Food Hydrocolloids. 85 (2018) 331-342.
482 [32] M.C. Ralet, V. Dronnet, H.C. Buchholt, J.F. Thibault, Enzymatically and
483 chemically de-esterified lime pectins: Characterisation, polyelectrolyte behaviour
484 and calcium binding properties, Carbohydr. Res. 336 (2001) 117-125.
485 [33] D.N. Sila, S. Van Buggenhout, T. Duvetter, I. Fraeye, A. De Roeck, A. Van Loey,
486 M. Hendrickx, Pectins in processed fruits and vegetables: Part II—Structure–
487 function relationships. Compr. Rev. Food Sci. Food Saf. 8(2) (2009) 86-104.
488 [34] S.H.E. Verkempinck, C. Kyomugasho, L. Salvia-Trujillo, S. Denis, M. Bourgeois,
489 A.M. Van Loey, M.E. Hendrickx, T. Grauwet, Emulsion stabilizing properties of
490 citrus pectin and its interactions with conventional emulsifiers in oil-in-water
491 emulsions, Food Hydrocolloids. 85 (2018) 144-157.
492 [35] Y. Tan, Z. Zhang, J.M. Mundo, D.J. McClements, Factors impacting lipid digestion
493 and nutraceutical bioaccessibility assessed by standardized gastrointestinal model
23
494 (INFOGEST): Emulsifier type, Food Res. Int. 137 (2020) 109739.
495 [36] K. Yao, D.J. McClements, C. Yan, J. Xiao, H. Liu, Z. Chen, X. Liu, In vitro and in
496 vivo study of the enhancement of carotenoid bioavailability in vegetables using
497 excipient nanoemulsions: Impact of lipid content, Food Res. Int. 141 (2021)
498 110162.
499 [37] S. Warnakulasuriya, P.K. Pillai, A.K. Stone, M.T. Nickerson, Effect of the degree
500 of esterification and blockiness on the complex coacervation of pea protein isolate
501 and commercial pectic polysaccharides, Food Chem. 264 (2018) 180-188.
502 [38] Y. Lan, J.B. Ohm, B. Chen, J. Rao, Phase behavior, thermodynamic and
503 microstructure of concentrated pea protein isolate-pectin mixture: Effect of pH,
504 biopolymer ratio and pectin charge density, Food Hydrocolloids. 101 (2020)
505 105556.
506 [39] J. Chen, R.H. Liang, L. Wei, S. J. Luo, C.M. Liu, S.S. Wu, et al. Extraction of
507 pectin from Premna microphylla turcz leaves and its physicochemical properties,
508 Carbohydr. Polym. 102(1) (2014) 376-384.
509 [40] Z. Zhang, M. Nie, Y. Xiao, L. Zhu, R. Gao, C. Zhou, Z. Dai, Positive effects of
510 ultrasound pretreatment on the bioaccessibility and cellular uptake of bioactive
511 compounds from broccoli: Effect on cell wall, cellular matrix and
512 digesta, LWT. 149 (2021) 112052.
513 [41] R. Zhang, G. Chen, B. Yang, Y. Wu, M. Du, J. Kan, Insights into the stability of
514 carotenoids and capsaicinoids in water-based or oil-based chili systems at different
515 processing treatments, Food Chem. 342 (2021) 128308.
24
516 [42] T.A.J. Verrijssen, S.H.E. Verkempinck, S. Christiaens, A.M. Van Loey, M. E.
517 Hendrickx, The effect of pectin on in vitro β-carotene bioaccessibility and lipid
518 digestion in low fat emulsions, Food Hydrocolloids. 49 (2015) 73-81.
519 [43] M. Iddir, J.F.P. Yaruro, Y. Larondelle, T. Bohn. Gastric lipase can significantly
520 increase lipolysis and carotenoid bioaccessibility from plant food matrices in the
521 harmonized INFOGEST static in vitro digestion model, Food Funct. 12(19) (2021)
522 9043-9053.
523 [44] K. Alba, V. Kontogiorgos, Pectin at the oil-water interface: Relationship of
524 molecular composition and structure to functionality, Food Hydrocolloids. 68
525 (2017) 211-218.
526 [45] D. A. Mendez, M. J. Fabra, A. Martínez-Abad, Μ. Μartínez-Sanz, M. Gorria, A.
527 López-Rubio, Understanding the different emulsification mechanisms of pectin:
528 Comparison between watermelon rind and two commercial pectin sources, Food
529 Hydrocolloids. 120 (2021) 106957.
530 [46] H. Niu, X. Chen, T. Luo, H. Chen, X. Fu, Relationships between the behavior of
531 three different sources of pectin at the oil-water interface and the stability of the
532 emulsion, Food Hydrocolloids. 128 (2022) 107566.
533 [47] L.M. de Souza Mesquita, B.V. Neves, L.P. Pisani, V. V. de Rosso, Mayonnaise as
534 a model food for improving the bioaccessibility of carotenoids from Bactris
535 gasipaes fruits, LWT. 122 (2020) 109022.
536 [48] L. Salvia-Trujillo, S.H.E. Verkempinck, X. Zhang, A.M. Van Loey, T. Grauwet,
537 M.E. Hendrickx, Comparative study on lipid digestion and carotenoid
25
538 bioaccessibility of emulsions, nanoemulsions and vegetable-based in situ
26
540 Figure captions
541 Fig. 1 Fourier transform infrared spectroscopy (FT-IR) of pectic polymers (A).
542 Composition and content of monosaccharides of pectic polymers (B). HG, RG-I
543 and RG are the abbreviations of homogalacturonan, rhamnogalacturonan-I and
544 rhamnogalacturonan, respectively.
545 Fig. 2 Hypothetical depiction of the pectic polymers structure. HG, RG-I and RG are
546 the abbreviations of homogalacturonan, rhamnogalacturonan-I and
548 Fig. 3 Pearson correlation analysis of pectic polymer structure and carotenoid
549 bioaccessibility. A positive and negative correlation between these indicators is
550 denoted by the colors blue and red, respectively. Higher color intensity represents
551 the higher correlation coefficient.
552 Fig. 4 ζ-potential (A) and apparent viscosity (B) of different systems during digestion.
553 Simulated systems containing β-carotene, β-carotene with HG, RG-I or RG were
554 abbreviated as SS, SS-HG, SS-RG-I, SS-RG, respectively. Different letters
555 indicate significant differences (P<0.05) among different systems in the same
556 digestion phase.
557 Fig. 5 β-carotene retention ratio and β-carotene bioaccessibility of different systems.
560 indicate statistically significant variations (P<0.05) among the systems during the
561 same digestion phase.
27
562 Fig. 6 Proposed mechanisms explaining the effects of pectic polymers on carotenoid
563 bioaccessibility. HG, RG-I and RG are the abbreviations of homogalacturonan,
564 rhamnogalacturonan-I and rhamnogalacturonan, respectively.
28
Revised manuscript (with changes marked)
1 Effects of pectin structure on carotenoid bioaccessibility in

2 simulated juice model
3
4 Jianing Liua,b, Jinfeng Bia,*, Xuan Liua,*, Dazhi Liua,c, Vincenzo Foglianob, Matthijs
5 Dekkerb, Ruud Verkerkb
a
7 Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences
8 (CAAS), Key Laboratory of Agro-Products Processing, Ministry of Agriculture and
9 Rural Affairs, Beijing 100193, China
b
10 Food Quality and Design Group, Wageningen University & Research, Bornse
c
12 Laboratory of Food Chemistry, Wageningen University & Research, Bornse
14
15
16
*
17 Corresponding author.
18 Tel.:+86-1062812584.
19 E-mail address: bijinfeng2010@163.com and liuxuancaas@126.com.
20 ADD: No. 2, Yuanmingyuan West Road, Haidian District, Beijing, 100193, China.
21
1
22 Abstract
23 The impact of pectin structure on carotenoid bioaccessibility is still uncertain. This
24 study aims to investigate how the different pectic polymers affected the bioaccessibility
25 of carotenoids in a simulated juice model during static in vitro digestion. This study
26 includes homogalacturonan (HG), which is a linear pectic polymer,
27 rhamnogalacturonan-I (RG-I), which is a branched pectic polymer, and
28 rhamnogalacturonan (RG), which is a diverse pectic polymer rich in RG-I,
29 rhamnogalacturonan-II (RG-II), and xylogalacturonan domains. Juice models without
30 pectin had the highest carotenoid bioaccessibility, suggesting pectin has negative
31 effects on carotenoid bioaccessibility. During the intestinal phase, systems with HG
32 showed the highest viscosity, followed by systems with RG and systems with RG-I.
33 Systems with RG-I had lower carotenoid bioaccessibility than systems with HG and
34 RG-II. Both the percentage of RG-I and the average side chain length of RG-I had
35 negative correlations with carotenoid bioaccessibility. RG-I side chains with more
36 arabinose and/or galactose might cause lower carotenoid bioaccessibility in this juice
37 model system.
38 Keywords: β-carotene bioaccessibility; Pectic polymers; Homogalacturonan;
39 Rhamnogalacturonan-I; Rhamnogalacturonan-II; Digestive fluids
2
40 Introduction
41 Pectin is a polysaccharide found in plant cell walls, predominantly consisting of
42 1,4-linked-α-D-galacturonic acid units. The primary structural domains of pectin are
43 homogalacturonan (HG), rhamnogalacturonan-I (RG-I), rhamnogalacturonan-II (RG-
44 II), and xylogalacturonan (XGA). HG makes up approximately 65% of the pectin
45 molecule, and it contains a linear chain of galacturonic acid (GalA) with methyl-
46 esterified carboxyl groups. RG-I is the second most abundant pectic polymer (i.e. 20-
47 35% of pectins), which comprises a backbone of repeating GalA and rhamnose
48 disaccharides with arabinan, galactan, and/or arabinogalactan side chains. The most
49 structurally complex pectin, RG-II, makes up about 10% of pectin. The backbone of
50 RG-II is made up of HG, with intricate side chains including simple sugars that are
51 connected to the GalA. XGA is an HG substituted at O-3 with a β-linked xylose, and it
52 is found in pectin in relatively small proportions (<7%) [1,2].
53 Different structural characteristics of pectic polymers contribute to their different
54 functional properties and bioactivities [3,4]. Many of pectin's functional characteristics
55 are influenced by the quantity of esterified GalA residues and their distribution in HG.
56 When methyl-ester groups are removed from HG, it becomes calcium cross-linkable,
57 allowing the formation of supramolecular assemblies and gels [5]. Furthermore, earlier
58 research has shown that cooperative cation binding to pectin requires at least 6 to 10
59 consecutive non-methyl-esterified GalA units [6,7]. RG-I pectin is heterogeneous due
60 to its variable glycosyl linkages, monosaccharide composition of side chains, chain
61 lengths, and distribution. Ferulic acid groups alter the molecular weight in the cell wall
3
62 and lead to poor pectin gelation properties by promoting the cross-linking of neutral
63 sugars in RG-I through ferulic bridges [8]. Recently, the research focused on the
64 importance of the pectin hairy region. Pectin RG-I portion showed improved
65 bioactivities, including inhibition of cell migration, and immunological and prebiotic
66 activities [9,10]. In vitro tests with human colon adenocarcinoma (Caco-2) cells showed
67 that RG-II had promisingly strong anti-cancer proliferation activities [11].
68 During the upper gastrointestinal stage, pectin may affect the digestion and
69 absorption of lipophilic carotenoids, by inhibiting lipid digestion [12]. Compared with
70 high pectin concentration, low pectin concentration favored the binding of bile salt and
71 carotenoid micellization [13]. The degree of lipid digestion decreased with an
72 increasing degree of pectin methyl-esterification [14]. Several studies have reported
73 carotenoid bioaccessibility and pectin structural and functional characteristics
74 (galacturonic acid concentration, degree of methyl-esterification and acetylation, and
75 emulsifying activity) of juices are related [15,16], however, the molecular mechanism
76 underpinning this relationship is still uncertain. It is possible that specific pectic
77 polymers are actually responsible for the reduction of carotenoid bioaccessibility
78 [12,16]. Therefore, the aim of this study was to investigate the influences of different
79 pectic polymers on carotenoid bioaccessibility of simulated juice models using static in
80 vitro digestion. In the present study, the pectic polymers include HG domain-rich pectic
81 polymer, RG-I region-rich pectic polymer, and rhamnogalacturonan (RG) pectic
82 polymer, which is more diverse with RG-I, RG-II and XGA domains. The simulated
83 juice models were applied to directly understand how pectin polymers influences β-
4
84 carotene digestion. Viscosity and ζ-potential of digestive fluids, β-carotene retention
85 and β-carotene bioaccessibility were recorded, to gain better knowledge of the digestive
86 process.
87
88 1. Materials and methods
89 2.1 Materials
90 Polygalacturonic acid (citrus pectin, ~5% methylation), rhamnogalacturonan I
91 (potato), and rhamnogalacturonan (soybean) were purchased from Megazyme (Bray,
92 Ireland). Polygalacturonic acid is seen as an HG domain-rich pectic polymer.
93 Rhamnogalacturonan I is considered to be an RG-I region-rich pectic polymer.
94 Rhamnogalacturonan is considered a more diverse pectic polymer rich in RG-I, RG-II
95 and XGA domains. Porcine gastric mucin, pepsin from porcine gastric mucosa, lipase
96 from porcine pancreas, β-carotene (≥ 93%), β-carotene (HPLC grade) and butylated
97 hydroxytoluene (BHT) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
98
99 2.2 Methods
100 2.2.1 Juice model system preparation
101 The juice model system was prepared according to our previous study, with minor
102 modifications [17]. β-carotene (30 mg) in 100 mL acetic acid-sodium acetate buffer (0.2
103 M, pH=6) with 0.2% corn oil (w/w) was dispersed using a high-speed dispersion mixer
104 (T25 Ultra-TURRAX, IKA, Germany) at 14,000 rpm for 40 min. Subsequently, HG
105 (polygalacturonic acid), RG-I (rhamnogalacturonan I), and RG (rhamnogalacturonan),
5
106 at a proportion of 1% (w/v), were added to the β-carotene solution, and fully mixed
107 using the Ultra-TURRAX at 7,000 rpm for 10 min. The systems were then adjusted to
108 pH 6 and homogenized at 250 MPa (SPCH-EP-IC-16-30, Homogenising Systems Ltd,
109 London, UK). Simulated systems containing β-carotene, β-carotene with HG, RG-I, or
110 RG were abbreviated as SS, SS-HG, SS-RG-I, and SS-RG, respectively (Table 1).
111
112 2.2.2 Structural characterization of pectic polymers
113 2.2.2.1 Fourier transform infrared spectroscopy (FT-IR) analysis
114 The FT-IR spectra of pectic polymers were determined using an FT-IR
115 spectrometer (Tensor 27, Bruker, Germany) in the wavenumber region 4000~400 cm-1
116 at a resolution of 4 cm-1 and a cumulative scan of 64. Before FTIR analysis, pectic
117 polymers were pulverized and pressed into flake with KBr at a ratio of 1:100 (w/w).
118
119 2.2.2.2 Determination of galacturonic acid (GalA) and neutral sugar concentration
120 GalA and neutral sugar concentrations were performed according to the previous
121 study [16]. High-performance anion-exchange chromatography (HPAEC) was achieved
122 using ICS-3000 system (Dionex, Sunnyvale, CA, USA) equipped with a CarboPacTM
123 PA20 column and a pulsed amperometric detector. Monosaccharides including GalA,
124 galactose (Gal), arabinose (Ara), rhamnose (Rha), xylose (Xyl), and fucose (Fuc) were
125 used for identification and quantification.
126 To obtain information on the domains of the pectic polymers, five composition
127 ratios were calculated based on the mol% quantifiable of GalA and neutral sugars, with
6
128 some modifications [18,19]:
GalA+Rha
129 The linearity of pectic polymers = (1)
GalA−Rha
130 The percentage of HG (% HG) = (2)
GalA−Rha
131 The ratio of HG to RG-I backbone = (3)
2×Rha
2×Rha+Ara+Gal
132 The percentage of RG-I (% RG-I) = (4)
Ara+Gal
133 The average side chain length of RG-I = (5)
Rha
134
135 2.2.2.3 Determination of molar mass distribution
136 The molar mass distributions of pectins were determined using high-performance
137 size exclusion chromatography (HPSEC) coupled with multiangle laser light scattering
138 (MALLS) (Dawn Heleos II, Wyatt Technology, Santa Barbara, CA, USA), reflective
139 index (RI) detector (Optilab rEX, Wyatt Technology, Santa Barbara, CA, USA) and
140 ultraviolet (UV) detector (L-2400, Hitachi, Tokyo, Japan). The dn/dc value (refractive
141 index increment) was 0.146 mL/g. Pectins (10 mg) were dissolved in eluent (0.1 M
142 NaCl) overnight and filtrated through a 0.45 μm filter membrane before analysis.
143 Samples (100 μL) were injected into an OHpak SB-806M HQ column (8.0 × 300 mm,
144 Shodex, Tokyo, Japan). Elution was performed with 0.1 M NaCl solution at a flow rate
145 of 0.5 mL/min for 30 min and the column was kept at 35 °C. The polydispersity index
146 (PDI) was calculated as the ratio of the weight-average molar mass (Mw) to the number
147 average molar mass (Mn).
148
149 2.2.3 Static in vitro digestion
7
150 Juice model systems were subjected to an in vitro digestion that encompassed
151 simulated oral, gastric, and intestinal phases [20]. During intestine digestion stage, an
152 automatic titration facility (Metrohm, USA, Inc.) was utilized to maintain pH at 7.0 by
153 adding 0.25 M NaOH solution over 2 h at 37°C. To stop the enzymatic reaction, samples
154 were placed on ice after each digestion phase.
155
156 2.2.4 Digestive fluid characteristics
157 2.2.4.1 Viscosity property of the digestive fluids
158 The viscosity of the digestive fluids at a shear rate of 50 s-1 was measured by using
159 a Physica MCR 301 rheometer (Anton Paar, Graz, Austria). The parallel plate geometry
160 had a 40 mm diameter and the gap size was 1 mm.
161
162 2.2.4.2 ζ-potential of the digestive fluids
163 The ζ-potential values of the initial systems (pectin solutions with β-carotene) and
164 their digestive fluids during digestion were measured by using a Zetasizer Nano ZS
165 analyzer (Malvern Instruments Ltd., Malvern, UK).
166
167 2.2.5 β-carotene bioaccessibility and retention ratio
168 After the above digestion process, the raw intestinal digesta was centrifuged at
169 8000 rpm (8801×g) and 4 °C for 1 h. β-carotene bioaccessibility was calculated as
170 follows:
171 β-carotene bioaccessibility (%)=(Cmicelle/Craw digesta) ×100 (6)
8
172 where Cmicelle and Craw digesta are the contents of β-carotene in the micelle fraction and
173 the raw digesta, respectively.
174 The β-carotene retention ratio during each digestion phase was calculated as
175 follows:
176 β-carotene retention ratio (%)=Cdigestion/Cinitial×100 (7)
177 where Cdigestion and Cinitial are the β-carotene concentrations (μg/mL) in different systems
178 during each digestion phase and in the initial system, respectively.
179
180 2.2.6 Extraction and determination of β-carotene
181 A previous procedure was used to extract and quantify β-carotene [21]. Samples
182 were separated and analyzed on high-performance liquid chromatography (HPLC)
183 system with a reversed-phase C30-column (250 mm × 4.6 mm, 5 μm; YMC Europe,
184 Dinslaken, Germany), a binary HPLC pump (1525, Waters, Milford, MA, USA) and a
185 photodiode array detector (2998, Waters, Milford, MA, USA). The gradient program,
186 detection wavelength, flow rate of mobile phase were performed according to a
187 previous procedure [16].
188
189 2.2.7 Statistics
190 The experiments were conducted in triplicate, and each sample was analyzed three
191 times to ensure accuracy and reproducibility. All data were presented as mean ±
192 standard deviation. Analysis of variance (ANOVA) followed by a Tukey test was used
193 to determine significant differences using R software (Version 4.2.0). The Pearson
9
194 correlation analysis was carried out by the “corrplot” package in R software.
195
196 2. Results and discussion
197 2.1 Structural characterization of pectin polymers
198 3.1.1 Functional groups of pectic polymers
199 As shown in Fig. 1A, the following were the main characteristics of the absorption
200 peaks: (i) The absorption peak at ≈ 3364 cm-1 was attributed to O-H stretching vibration,
201 and the peak at ≈ 2936 cm-1 was caused by C-H stretching of CH2 groups; (ii) the peaks
202 at ≈ 1745 cm-1 and 1608 cm-1 corresponded to the C=O stretching vibration of methyl-
203 esterified carboxyl groups and C=O asymmetrical stretching vibration of free carboxyl,
204 respectively; (iii) the peak at ≈ 1418 cm-1 was ascribed to a symmetric carboxylate
205 stretch [22,23]. The ratio of peak area at 1745 cm-1 (related to methyl ester carbonyl
206 group stretching) to the sum of the peak areas at 1745 cm-1 and 1608 cm-1 (related to
207 carboxylate group stretching) was calculated as the degree of methyl-esterification (DM)
208 [24,25]. The calculated DM of HG and RG-I were 8% and 4%, respectively. However,
209 the absorption area of RG at 1745 cm-1 was not detected, which suggested that methyl-
210 esters were not present in the RG chain. The variation of the DM observed could be
211 attributed to the different extraction methods applied.
212
213 3.1.2 Monosaccharide content and composition ratios of pectic polymers
214 As shown in Fig. 1B, the GalA content of HG was the highest (94.1%), followed
215 by RG-I (70.9%) and RG (50.9%). HG is composed of 94% GalA and 6% other
10
216 monosaccharides, such as 3.9% Gal and 1.5% Rha. RG-I comprises 70.9% GalA,
217 followed by 19.8% Gal, 6.3% Rha, and 1.8% Ara that constitute the RG-I region. RG
218 is a more diverse pectic polymer compared to HG and RG-I, as evidenced by its
219 composition of 50.9% GalA, 16.9% Xyl, and 13.1% Fuc. Individual monosaccharide
220 contents are not sufficient to gain insight into the pectin chains. With the help of the
221 ratios between the composing monosaccharides, the overall structure of pectin chains
222 becomes more visible.
223 As shown in Table 2, compared to the other two pectic polymers, HG is composed
224 of more linear/less branched pectin evidenced by the higher linearity of pectic
225 polymers, % HG and the ratio of HG to RG-I backbone. The higher linearity of HG
226 makes it potential to have higher Ca2+-binding capability, due to the higher possibility
227 of consecutive non-methyl-esterified GalA residues. RG-I is composed of a more
228 branched RG-I domain according to higher % RG-I and average side chain length of
229 RG-I. Pectin polymers rich in RG-I side chains have better emulsion stability by
230 forming a hydrated layer [26]. RG is a pectic polymer with more diverse domains,
231 which consists of 26.87% RG-I and approximately 13.14% RG-II side chains and 16.94%
232 XGA side chains. The percentage of the RG-II and XGA backbones is unclear, though,
233 as both of their backbones are HG. Based on the obtained structural characteristics, the
234 structures of the pectic polymers are visualized as hypothetical model structures
235 presented in Fig. 2. These distinct monosaccharide compositions among different pectic
236 polymers make it possible for further investigation of the effects of pectic polymers on
237 carotenoid bioaccessibility.
11
238
239 3.1.3 Molar mass distribution of pectic polymers
240 Mw and PDI values are presented in Table 2, and a higher PDI represents a wider
241 molecular mass distribution. RG showed the highest Mw, followed by RG-I and HG.
242 HG showed the lowest PDI values compared to RG and RG-I. The rich side chains of
243 RG-I and RG might contribute to their higher Mw and wider molecular mass distribution.
244 According to correlation analysis, the linearity of pectin had a negative correlation with
245 Mw, indicating that a higher degree of rhamnification might correspond to a larger Mw
246 of the polysaccharide (Fig. 3). Many pectin properties are linked to its Mw, and high
247 molecular mass polymers generally contribute to promising functionalities, such as
248 emulsifying and emulsion stabilizing abilities [27]. In most cases, higher Mw
249 macromolecules obstruct fluid motion, resulting in higher viscosity solutions [28]. It
250 should be noted that other factors, such as pectin sources and pectin fractions, might
251 also affect the viscosity of pectins [29,30]. Decreasing Mw might reduce the maximum
252 amount of Ca2+ that pectin can bind [31], however, the calcium binding ability also
253 depends on the degree and distribution pattern of methyl-ester groups among HG region
254 [32].
255
256 3.2 Physical characteristics of digestive fluids
257 3.2.1 ζ-potential of digestive fluids
258 Different physical indicators were estimated to investigate the effects of pectic
259 polymers on digestion process. Generally, changes in the ζ-potential of particles can
12
260 influence their electrostatic repulsion or attraction, which in turn can affect the stability
261 and behavior of the systems during digestion. As seen in Fig. 4A, initial ζ-potential
262 values of SS-HG and SS-RG were lower than those of SS and SS-RG-I, which
263 suggested that SS-HG and SS-RG were enveloped by more negatively charged
264 substances. Typically, an increased GalA content in pectin results in a higher negative
265 charge [33]. Additionally, more demethylesterified pectin can carry more negative
266 charges [34]. During the oral phase, the ζ-potential value of SS was the highest,
267 followed by SS-RG, SS-RG-I and SS-HG. The ζ-potential values increased from the
268 initial phase to the gastric phase, while decreased in the intestinal phase. The highly
269 acidic conditions, which cause the protein-coated droplets to be positive, and the
270 presence of anionic mucin, which binds to the cationic protein surfaces, are both related
271 to the increase in ζ-potential during the gastric phase [35,36]. Specifically, GalA, the
272 degree of methyl-esterification and the distribution of methyl esters of pectin influence
273 their interactions with protein [37]. Moreover, the pectin surface charge decreased with
274 the decreasing pH, and at pH 2, the ζ-potential of pectin is close to zero [38]. The
275 amount of the negative charge on the backbone of pectin decreased as a result of
276 protonation (a drop in pH). The fact that the ζ-potential values changed from their initial
277 positive values to more negative ones during the intestinal phase could be attributed to
278 a number of negatively charged species, including free fatty acids released during
279 lipolysis, intricate colloidal structures, undigested oil, and anionic polymers [14].
280
281 3.2.2 Apparent viscosity of digestive fluids
13
282 The apparent viscosity of digestive fluids is given in Fig. 4B. The physical feature
283 of apparent viscosity is linked to soluble dietary fibers, particularly pectic
284 polysaccharides. Initial SS-HG and SS-RG showed higher viscosity than SS-RG-I.
285 Generally, pectin with a higher Mw has a higher viscosity [28]. Moreover, HG pectin
286 contributed greatly to the viscosity of pectin [39]. There was a significant negative
287 correlation between the initial viscosity of the systems and the RG-I percentage (Fig.
288 3). Due to the neutral sugar side chains, solutions containing high contents of RG-I
289 and/or RG-II pectin usually have lower viscosity values than solutions with high GalA
290 content [39]. From the initial phase to the gastric phase, the viscosity of the four systems
291 all decreased, which might be caused by the dilution effect of digestive fluids. These
292 results are consistent with those obtained with broccoli-digested fractions, where
293 viscosity decreased from the gastric phase to the intestinal phase [40]. During the
294 intestinal phase, SS-HG showed the highest viscosity, followed by SS-RG, SS-RG-I
295 and SS. A negative correlation has been found between the HG to RG-I ratio and the
296 viscosity of the systems during the intestinal phase. The interactions between pectic
297 polymers and the digestive fluids might also contribute to the results, especially the
298 crosslinking between pectin and digestive enzymes.
299
300 3.3 β-carotene retention ratio during digestion phases
301 As seen in Fig. 5, in the oral phase, the β-carotene retention ratio of SS-RG was
302 lower than that of SS, while in the gastric phase, the β-carotene retention ratio of SS-
303 RG was the highest compared with other systems. This suggested that SS-RG might be
14
304 more stable under acidic conditions. At the intestinal phase, the β-carotene retention
305 ratios of SS-HG, SS-RG-I, and SS-RG had no significant difference. Carotenoids are
306 isoprenoid pigments with a polyene backbone that contains a variable number of
307 conjugated double bonds, therefore, they are prone to be oxidized. A previous study
308 showed that the stability of carotenoids at different processing can be significantly
309 improved in an oil-based system, compared with a water-based juice system [41]. In
310 our study, the carotenoids were in a homogenized juice model system, which is similar
311 to a water-based juice system, therefore, carotenoid stability might be affected by high-
312 pressure homogenization to some extent. A negative correlation was observed between
313 the β-carotene retention ratio during the intestinal phase and the ζ-potential values of
314 initial systems. This suggests that a higher negative charge could potentially enhance
315 the delivery of carotenoids during the intestinal stage. Additionally, a negative
316 correlation was found between the β-carotene retention ratio and the viscosity of
317 digestive fluids during the intestinal phase. Therefore, the pectin-induced viscosity
318 might have effects on the β-carotene stability during the intestinal stage.
319
320 3.4 β-carotene bioaccessibility
321 Carotenoids need to be released from the food matrix, solubilized into the lipid
322 phase, and incorporated into mixed micelles in order to be absorbed via the human
323 intestinal epithelium [12]. The levels of carotenoids available for intestinal absorption,
324 specifically the carotenoids in mixed micelles, are referred to as bioaccessible
15
325 carotenoids. SS had the highest β-carotene bioaccessibility compared to other systems
326 (SS-HG, SS-RG-I and SS-RG), which signified that the presence of pectin could reduce
327 the bioaccessibility carotenoids, thus limiting their potential health effects. Similar
328 observations were also found by Cervantes-Paz et.al [13], in which high pectin
329 concentration decreased carotenoid micellization. Pectin can increase the viscosity of
330 digestive fluids, allowing digesta to last longer in the gastrointestinal phase while
331 simultaneously reducing the movement of substrates and digestive enzymes (eg., pepsin
332 and lipase), decreasing lipase activity and micelle generation [42]. Moreover, different
333 pectic polymers caused different influences on carotenoid bioaccessibility. SS-HG and
334 SS-RG had similar β-carotene bioaccessibility, while SS-RG-I had a smaller β-carotene
335 bioaccessibility than those of SS-HG and SS-RG. Furthermore, both % RG-I and the
336 average side chain length of RG-I had negative correlations with carotenoid
337 bioaccessibility. Therefore, it was deduced that more RG-I side chains with Ara and/or
338 Gal might cause lower carotenoid bioaccessibility in this juice model system. These
339 results are consistent with those obtained with carrot juice, where higher emulsifying
340 properties caused a lower value of total carotenoid bioaccessibility [16]. The proposed
341 mechanisms of the effects of pectic polymers on carotenoid bioaccessibility are shown
342 in Fig. 6. The lipid hydrolysis produces monoacylglycerols and free fatty acids, which
343 together with bile salts contribute to the formation of mixed micelles. In this context,
344 lipase can greatly increase lipolysis and carotenoid bioaccessibility [43]. Pectin
345 segments with enriched RG-I/neutral sugar side chains contribute to better emulsifying
346 properties [44,45]. Pectin with better emulsification properties might be able to form
16
347 thicker hydrated layers at the oil-water interface and inhibit lipase to the surface,
348 resulting in low carotenoid bioaccessibility. A recent study based on molecular
349 dynamics simulations also revealed that RG-I and RG-II domains were adsorbed on the
350 oil-water interface in a folded form, creating a thick viscoelastic film while the HG
351 domain was tiled on the interface [46].
352 It should also be mentioned that in real fruit and vegetable systems, RG-I side
353 chains could also be cross-linked to xylans, xyloglucans, lignins, and proteins [5]. The
354 above-mentioned cross-linking among pectic polymers can also influence carotenoid
355 bioaccessibility, but this is not considered in the juice model system in this study.
356 Despite the absence of explicit localization obstacles of carotenoids in model systems,
357 β-carotene bioaccessibility was still comparatively low, ranging from 7.97% to 14.55%.
358 It implies that lipids are important in enhancing β-carotene bioaccessibility since dietary
359 lipids could stimulate bile salt secretion and increase the number of carotenoids carried
360 in micelles. Furthermore, other consumption forms of food products might be helpful
361 in increasing carotenoid bioaccessibility. Making fruit and vegetable-based emulsions
362 has been noted as a promising method to increase carotenoid bioaccessibility by
363 researchers [47,48]. Therefore, further research might focus on the effects of pectic
364 polymers on carotenoid bioaccessibility in model systems with the addition of more
365 lipids.
366
367 4. Conclusions
368 The simulated juice models were helpful to understand the effects of different
17
369 pectic polymers on carotenoid bioaccessibility. The ζ-potential values of all the systems
370 increased from the initial phase to the gastric phase, while decreased in the intestinal
371 phase. When compared to the initial ζ-potential values, ζ-potential became more
372 negative during the intestinal phase. During the intestinal phase, SS-HG showed the
373 highest viscosity, followed by SS-RG, SS-RG-I and SS. SS-HG and SS-RG had similar
374 β-carotene bioaccessibility, while SS-RG-I had a lower β-carotene bioaccessibility than
375 those of SS-HG and SS-RG. Both % RG-I and the average side chain length of RG-I
376 had negative influences on carotenoid bioaccessibility.
377
378 Acknowledgements
379 This work was supported by the National Key Research and Development
380 Program of China (2022YFD1600505), the earmarked fund for China Agriculture
381 Research System (CARS-27), and the Joint PhD Program between Wageningen
382 University & Research (WUR) and Chinese Academy of Agricultural Sciences (CAAS)
383 (No. MOE11NL1A20151701N).
18
384 References
385 [1] D. Mohnen, Pectin structure and biosynthesis, Curr. Opin. Plant Biol. 11(3) (2008)
386 266-277.
387 [2] J. Zandleven, S.O. Sørensen, J. Harholt, G. Beldman, H.A. Schols, H.V. Scheller, &
388 A.J. Voragen, Xylogalacturonan exists in cell walls from various tissues of
389 Arabidopsis thaliana, Phytochemistry. 68(8) (2007) 1219-1226.
390 [3] B. Neckebroeck, S.H.E. Verkempinck, G. Vaes, K. Wouters, J. Magnee, M. E.
391 Hendrickx, et al. Advanced insight into the emulsifying and emulsion stabilizing
392 capacity of carrot pectin subdomains, Food Hydrocolloids, 102 (2020) 105594.
393 [4] P. M. Pieczywek, J. Ciesla, W. Płazinski, A. Zdunek, Aggregation and weak gel
394 formation by pectic polysaccharide homogalacturonan, Carbohydr. Polym. 256
395 (2021) 117566.
396 [5] W.G.T. Willats, L. Mccartney, W. Mackie, & J.P. Knox, Pectin: Cell biology and
397 prospects for functional analysis, Plant Mol. Biol. 47(1-2) (2001) 9-27.
398 [6] P.J.H. Daas, B. Boxma, A.M.C.P. Hopman, A.G.J. Voragen, H.A. Schols,
399 Nonesterified galacturonic acid sequence homology of pectins, Biopolymers. 58(1)
400 (2001) 1-8.
401 [7] G.A. Luzio, R.G. Cameron, Demethylation of a model homogalacturonan with the
402 salt-independent pectin methylesterase from citrus: Part II. structure–function
403 analysis, Carbohydr. Polym. 71(2) (2008) 300-309.
404 [8] A. Oosterveld, I.E. Pol, G. Beldman, A.G. Voragen, Isolation of feruloylated
405 arabinans and rhamnogalacturonans from sugar beet pulp and their gel forming
19
406 ability by oxidative cross-linking, Carbohydr. Polym. 44(1) (2001) 9-17.
407 [9] D. Wu, J. Zheng, G. Mao, W. Hu, X. Ye, R.J. Linhardt, et al. Rethinking the impact
408 of RG-I mainly from fruits and vegetables on dietary health, Crit. Rev. Food Sci.
409 Nutr. 60 (2020) 2938-2960.
410 [10] M.Y. Zhang, J. Cai, Preparation of branched RG-I-rich pectin from red dragon fruit
411 peel and the characterization of its probiotic properties, Carbohydr. Polym. (2023)
412 120144.
413 [11] L. Ai, Y. Chung, S. Lin, K. Lee, P. Lai, Y. Xia, et al. Active pectin fragments of
414 high in vitro antiproliferation activities toward human colon adenocarcinoma cells:
415 Rhamnogalacturonan II, Food Hydrocolloids. 83 (2018) 239-245.
416 [12] B. Cervantes-Paz, J.J. Ornelas-Paz, S. Ruiz-Cruz, C. Rios-Velasco, V. Ibarra-
417 Junquera, E.M. Yahia, et al. Effects of pectin on lipid digestion and possible
418 implications for carotenoid bioavailability during pre-absorptive stages: A review,
419 Food Res. Int. 99 (2017) 917-927.
420 [13] B. Cervantes-Paz, J.D.J. Ornelas-Paz, J.D. Pérez-Martínez, J. Reyes-Hernández,
421 P.B. Zamudio-Flores, C. Rios-Velasco, et al. Effect of pectin concentration and
422 properties on digestive events involved on micellarization of free and esterified
423 carotenoids, Food Hydrocolloids. 60 (2016) 580-588.
424 [14] M. Espinal-Ruiz, L.P. Restrepo-Sanchez, C.E. Narvaez-Cuenca, D.J. McClements,
425 Impact of pectin properties on lipid digestion under simulated gastrointestinal
426 conditions: Comparison of citrus and banana passion fruit (Passiflora tripartita var.
427 mollissima) pectins, Food Hydrocolloids. 52 (2016) 329-342.
20
428 [15] Y. Ding, X. Liu, J. Bi, X. Wu, X. Li, J. Liu, et al. Effects of pectin, sugar and pH
429 on the β-Carotene bioaccessibility in simulated juice systems, LWT--Food Sci.
430 Technol. 124 (2020) 109125.
431 [16] X. Liu, J. Liu, J. Bi, J. Yi, J. Peng, C. Ning, et al. Effects of high pressure
432 homogenization on pectin structural characteristics and carotenoid bioaccessibility
433 of carrot juice, Carbohydr. Polym. 203 (2019) 176-184.
434 [17] J. Liu, J. Bi, X. Liu, D. Liu, X. Wu, J. Lyu, et al. Effects of pectins and sugars on
435 β-carotene bioaccessibility in an in vitro simulated digestion model, J. Food
436 Compos. Anal. 91 (2020) 103537.
437 [18] S.E. Broxterman, P. Picouet, H.A. Schols, Acetylated pectins in raw and heat
438 processed carrots, Carbohydr. Polym. 177 (2017) 58-66.
439 [19] K. Houben, R.P. Jolie, I. Fraeye, A.M. Van Loey, M.E. Hendrickx, Comparative
440 study of the cell wall composition of broccoli, carrot, and tomato: Structural
441 characterization of the extractable pectins and hemicelluloses, Carbohydr. Res.
442 346(9) (2011) 1105-1111.
443 [20] J. Liu, D. Liu, J. Bi, X. Liu, Y. Lyu, R. Verkerk, et al. Micelle separation conditions
444 based on particle size strongly affect carotenoid bioaccessibility assessment from
445 juices after in vitro digestion, Food Res. Int. 151 (2022) 110891.
446 [21] G. Knockaert, L. Lemmens, S. Van Buggenhout, M. Hendrickx, A. Van Loey,
447 Changes in β-carotene bioaccessibility and concentration during processing of
448 carrot puree, Food Chem. 133(1) (2012) 60-67.
449 [22] M. Kazemi, F. Khodaiyan, S.S. Hosseini, Utilization of food processing wastes of
21
450 eggplant as a high potential pectin source and characterization of extracted pectin,
451 Food Chem. 294 (2019) 339-346.
452 [23] J.S. Yang, T.H. Mu, M.M. Ma, Extraction, structure, and emulsifying properties of
453 pectin from potato pulp, Food Chem. 244 (2018) 197-205.
454 [24] G.D. Manrique, F.M. Lajolo, FT-IR spectroscopy as a tool for measuring degree
455 of methyl esterification in pectins isolated from ripening papaya fruit. Postharvest
456 Biol. Technol. 25(1) (2002) 99-107.
457 [25] C. Kyomugasho, S. Christiaens, A. Shpigelman, A.M. Van Loey, M.E. Hendrickx,
458 FT-IR spectroscopy, a reliable method for routine analysis of the degree of
459 methylesterification of pectin in different fruit-and vegetable-based matrices. Food
460 Chem. 176 (2015) 82-90.
461 [26] T. Funami, M. Nakauma, S. Ishihara, R. Tanaka, T. Inoue, G.O. Phillips, Structural
462 modifications of sugar beet pectin and the relationship of structure to
463 functionality, Food Hydrocolloids. 25(2) (2011) 221-229.
464 [27] B. Neckebroeck, S.H.E. Verkempinck, T. Bernaerts, D. Verheyen, M.E. Hendrickx,
465 A. M. Van Loey, Investigating the role of the different molar mass fractions of a
466 pectin rich extract from onion towards its emulsifying and emulsion stabilizing
467 potential, Food Hydrocolloids. 117 (2021) 106735.
468 [28] L. Wan, Q. Chen, M. Huang, F. Liu, S. Pan, Physiochemical, rheological and
469 emulsifying properties of low methoxyl pectin prepared by high hydrostatic
470 pressure-assisted enzymatic, conventional enzymatic, and alkaline de-
471 esterification: A comparison study, Food Hydrocolloids. 93 (2019) 146-155.
22
472 [29] J. Mierczyńska, J. Cybulska, P.M. Pieczywek, A. Zdunek, Effect of storage on
473 rheology of water-soluble, chelate-soluble and diluted alkali-soluble pectin in
474 carrot cell walls, Food Bioprocess Technol. 8 (2015) 171-180.
475 [30] J. Mierczyńska, J. Cybulska, A. Zdunek, Rheological and chemical properties of
476 pectin enriched fractions from different sources extracted with citric
477 acid, Carbohydr. Polym. 156 (2017) 443-451.
478 [31] C. Kyomugasho, C. Munyensanga, M. Celus, K. Dewettinck, A.M. Van Loey, T.
479 Grauwet, M.E. Hendrickx, Molar mass influence on pectin-Ca2+ adsorption
480 capacity, interaction energy and associated functionality: Gel microstructure and
481 stiffness, Food Hydrocolloids. 85 (2018) 331-342.
482 [32] M.C. Ralet, V. Dronnet, H.C. Buchholt, J.F. Thibault, Enzymatically and
483 chemically de-esterified lime pectins: Characterisation, polyelectrolyte behaviour
484 and calcium binding properties, Carbohydr. Res. 336 (2001) 117-125.
485 [33] D.N. Sila, S. Van Buggenhout, T. Duvetter, I. Fraeye, A. De Roeck, A. Van Loey,
486 M. Hendrickx, Pectins in processed fruits and vegetables: Part II—Structure–
487 function relationships. Compr. Rev. Food Sci. Food Saf. 8(2) (2009) 86-104.
488 [34] S.H.E. Verkempinck, C. Kyomugasho, L. Salvia-Trujillo, S. Denis, M. Bourgeois,
489 A.M. Van Loey, M.E. Hendrickx, T. Grauwet, Emulsion stabilizing properties of
490 citrus pectin and its interactions with conventional emulsifiers in oil-in-water
492 [35] Y. Tan, Z. Zhang, J.M. Mundo, D.J. McClements, Factors impacting lipid digestion
493 and nutraceutical bioaccessibility assessed by standardized gastrointestinal model
23
494 (INFOGEST): Emulsifier type, Food Res. Int. 137 (2020) 109739.
495 [36] K. Yao, D.J. McClements, C. Yan, J. Xiao, H. Liu, Z. Chen, X. Liu, In vitro and in
496 vivo study of the enhancement of carotenoid bioavailability in vegetables using
497 excipient nanoemulsions: Impact of lipid content, Food Res. Int. 141 (2021)
498 110162.
499 [37] S. Warnakulasuriya, P.K. Pillai, A.K. Stone, M.T. Nickerson, Effect of the degree
500 of esterification and blockiness on the complex coacervation of pea protein isolate
501 and commercial pectic polysaccharides, Food Chem. 264 (2018) 180-188.
502 [38] Y. Lan, J.B. Ohm, B. Chen, J. Rao, Phase behavior, thermodynamic and
503 microstructure of concentrated pea protein isolate-pectin mixture: Effect of pH,
504 biopolymer ratio and pectin charge density, Food Hydrocolloids. 101 (2020)
505 105556.
506 [39] J. Chen, R.H. Liang, L. Wei, S. J. Luo, C.M. Liu, S.S. Wu, et al. Extraction of
507 pectin from Premna microphylla turcz leaves and its physicochemical properties,
508 Carbohydr. Polym. 102(1) (2014) 376-384.
509 [40] Z. Zhang, M. Nie, Y. Xiao, L. Zhu, R. Gao, C. Zhou, Z. Dai, Positive effects of
510 ultrasound pretreatment on the bioaccessibility and cellular uptake of bioactive
511 compounds from broccoli: Effect on cell wall, cellular matrix and
512 digesta, LWT. 149 (2021) 112052.
513 [41] R. Zhang, G. Chen, B. Yang, Y. Wu, M. Du, J. Kan, Insights into the stability of
514 carotenoids and capsaicinoids in water-based or oil-based chili systems at different
515 processing treatments, Food Chem. 342 (2021) 128308.
24
516 [42] T.A.J. Verrijssen, S.H.E. Verkempinck, S. Christiaens, A.M. Van Loey, M. E.
517 Hendrickx, The effect of pectin on in vitro β-carotene bioaccessibility and lipid
518 digestion in low fat emulsions, Food Hydrocolloids. 49 (2015) 73-81.
519 [43] M. Iddir, J.F.P. Yaruro, Y. Larondelle, T. Bohn. Gastric lipase can significantly
520 increase lipolysis and carotenoid bioaccessibility from plant food matrices in the
521 harmonized INFOGEST static in vitro digestion model, Food Funct. 12(19) (2021)
522 9043-9053.
523 [44] K. Alba, V. Kontogiorgos, Pectin at the oil-water interface: Relationship of
524 molecular composition and structure to functionality, Food Hydrocolloids. 68
525 (2017) 211-218.
526 [45] D. A. Mendez, M. J. Fabra, A. Martínez-Abad, Μ. Μartínez-Sanz, M. Gorria, A.
527 López-Rubio, Understanding the different emulsification mechanisms of pectin:
528 Comparison between watermelon rind and two commercial pectin sources, Food
529 Hydrocolloids. 120 (2021) 106957.
530 [46] H. Niu, X. Chen, T. Luo, H. Chen, X. Fu, Relationships between the behavior of
531 three different sources of pectin at the oil-water interface and the stability of the
532 emulsion, Food Hydrocolloids. 128 (2022) 107566.
533 [47] L.M. de Souza Mesquita, B.V. Neves, L.P. Pisani, V. V. de Rosso, Mayonnaise as
534 a model food for improving the bioaccessibility of carotenoids from Bactris
535 gasipaes fruits, LWT. 122 (2020) 109022.
536 [48] L. Salvia-Trujillo, S.H.E. Verkempinck, X. Zhang, A.M. Van Loey, T. Grauwet,
537 M.E. Hendrickx, Comparative study on lipid digestion and carotenoid
25
538 bioaccessibility of emulsions, nanoemulsions and vegetable-based in situ
26
540 Figure captions
541 Fig. 1 Fourier transform infrared spectroscopy (FT-IR) of pectic polymers (A).
542 Composition and content of monosaccharides of pectic polymers (B). HG, RG-I
543 and RG are the abbreviations of homogalacturonan, rhamnogalacturonan-I and
545 Fig. 2 Hypothetical depiction of the pectic polymers structure. HG, RG-I and RG are
546 the abbreviations of homogalacturonan, rhamnogalacturonan-I and
548 Fig. 3 Pearson correlation analysis of pectic polymer structure and carotenoid
549 bioaccessibility. A positive and negative correlation between these indicators is
550 denoted by the colors blue and red, respectively. Higher color intensity represents
551 the higher correlation coefficient.
552 Fig. 4 ζ-potential (A) and apparent viscosity (B) of different systems during digestion.
555 indicate significant differences (P<0.05) among different systems in the same
556 digestion phase.
557 Fig. 5 β-carotene retention ratio and β-carotene bioaccessibility of different systems.
560 indicate statistically significant variations (P<0.05) among the systems during the
561 same digestion phase.
27
562 Fig. 6 Proposed mechanisms explaining the effects of pectic polymers on carotenoid
563 bioaccessibility. HG, RG-I and RG are the abbreviations of homogalacturonan,
564 rhamnogalacturonan-I and rhamnogalacturonan, respectively.
28
Highlights
Highlights
 Simulated models reveal links between pectin and carotenoid bioaccessibility.
 Carotenoid bioaccessibility was lower in rhamnogalacturonan-I system.
 Rhamnogalacturonan-I ratio is negatively linked with carotenoid bioaccessibility.
 Longer side chains of rhamnogalacturonan-I reduce carotenoid bioaccessibility.

Figure Click here to access/download;Figure;Figures.docx
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Table (Editable version) Click here to access/download;Table (Editable version);Tables.doc
Table 1 Juice model systems in this study.
Juice systems Abbreviations
β-carotene solution SS
β-carotene solution+ 1% polygalacturonic acid SS-HG
β-carotene solution+ 1% rhamnogalacturonan I SS-RG-I
β-carotene solution+ 1% rhamnogalacturonan SS-RG
Table 2 Structural characteristics of different pectic polymers.
Homogalacturonan (HG) Rhamnogalacturonan-I (RG-I) Rhamnogalacturonan (RG)
The linearity of pectic polymers 95.59 ± 0.06 77.21 ± 0.32 58.73 ± 0.16
The percentage of HG (% HG) 92.56 ± 0.22 64.62 ± 0.77 43.06 ± 0.20
The ratio of HG to RG-I backbone 30.56 ± 1.61 5.13 ± 0.24 2.75 ± 0.08
The percentage of RG-I (% RG-I) 7.27 ± 0.23 34.18 ± 0.86 26.87 ± 0.27
The average side chain length of RG-I 2.80 ± 0.09 3.43 ± 0.06 1.43 ± 0.04
Weight-average molar mass (Mw) (g/mol) 9.63 × 104 4.53 × 105 1.43 × 106
The polydispersity index (PDI) 2.15 ± 0.36 2.11 ± 0.04 3.07 ± 0.11
The results were expressed as mean ± standard deviation.

Ijbiomac D 23 13059 r1 Reviewer

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Ijbiomac D 23 13059 r1 Reviewer

Uploaded by

Copyright:

Available Formats

International Journal of Biological Macromolecules

Effects of pectin structure on carotenoid bioaccessibility in simulated juice model

Manuscript Number: IJBIOMAC-D-23-13059R1

Article Type: Research Paper

Section/Category: Carbohydrates, Natural Polyacids and Lignins

Keywords: β-carotene bioaccessibility; Pectic polymers; homogalacturonan

Response to Reviewer Comments

Manuscript Number: IJBIOMAC-D-23-13059

Title: Effects of pectin structure on carotenoid bioaccessibility in simulated juice

Journal: International Journal of Biological Macromolecules

Dear Prof. Corsaro,

been highlighted in red. Point-by-point responses to the reviewers’ comments are

listed below this letter.

Highlights: in the final highlight is it possible to indicate how RG-I affects

bioavailability? Positively/ negatively?

negatively affects carotenoid bioaccessibility. Due to the word limitation of each

you suggest - how RG-I causes lower bioavailability in the abstract?

homogalacturonan (HG), which is a linear pectic polymer, rhamnogalacturonan-I

(RG-I), which is a branched pectic polymer, and rhamnogalacturonan (RG), which is

a diverse pectic polymer rich in RG-I, rhamnogalacturonan-II (RG-II), and

carotenoid bioaccessibility in this juice model system.

Line 50 - what is the proportion of XGA?

in pectin in relatively small proportions (<7%).

Line 75 - could you add a reference?

References have been added in Line 78.

our study is to investigate the different pectin subdomains on carotenoid

bioaccessibility. Different sources contribute to the different pectin subdomains,

provide distinct pectin subdomains.

Line 90 - do you know DE or DM?

This polygalacturonic acid product contains approximately 5% methylation according

Line 102 - mixer rather than machine

“Machine” has been modified into “mixer” (Line 103).

We have added a table with sample information (Table 1).

the Materials & Methods (Line 163-164).

we have added this estimated DM values in Line 205-208.

carboxylate group stretching) was calculated as the degree of methyl-esterification

Lines 188 - 189 - some repetition

We have modified the sentence according to your comment (Line 190-191).

analyzed three times to ensure accuracy and reproducibility.

Could you add a reference?

an effective means to estimate the degree of methyl-esterification (DM) of pectin.

carboxylate group stretching) was calculated as DM [24,25].

different fruit-and vegetable-based matrices. Food Chem. 176 (2015) 82-90.

Line 242 - should it be rhamnification?

Yes, it is. We have now corrected the word (Line 245).

We have modified this into Fig. 3 (Line 246).

Line 255 - check your wording

We have modified this sentence (Line 258-259).

of pectic polymers on digestion process.

We have added more discussion regarding GalA content (Line 264-266).

Line 264-266: Typically, an increased GalA content in pectin results in a higher

negative charges [34].

Line 269 - "...pectin is close to zero.."

This sentence has been modified (Line 274).

figure as Fig. 5A and Fig. 5B according to your comment.

Line 320 - "... bioavailability compared to other systems..."

This sentence has been modified (Line 325).

Line 325-327: SS had the highest β-carotene bioaccessibility compared to other

we have removed this statement from the manuscript.

Line 354 - what are the units/ %?

We have added the unit (%) in Line 357.

Lines 357 - 358 - "...helpful in increasing ..."

Line 370 - "... SS-RG-I had lower ..."