FACULTY OF BIOSCIENCE ENGINEERING Interuniversity Programme Master of Science in Food Technology (IUPFOOD) Effect of structural modification of citrus and sugar beet pectins on emulsifying capacity Promoter: Prof. Dr. Ir. Marc Hendrickx Dissertation presented in fulfillment Co-promoter: Dr. Ir. Stefanie Christiaens of the requirements for the degree of Department for of Microbial and Molecular Systems Master of Science in Food Technology Centre for Food and Microbial Technology Gladys Kontoh September 2015
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FACULTY OF BIOSCIENCE ENGINEERING
Interuniversity Programme
Master of Science in Food Technology (IUPFOOD)
Effect of structural modification of citrus and sugar beet
pectins on emulsifying capacity
Promoter: Prof. Dr. Ir. Marc Hendrickx Dissertation presented in fulfillment
Co-promoter: Dr. Ir. Stefanie Christiaens of the requirements for the degree of
Department for of Microbial and Molecular Systems Master of Science in Food Technology
Centre for Food and Microbial Technology
Gladys Kontoh
September 2015
This dissertation is part of the examination and has not been corrected for eventual errors
after presentation. Use as a reference is only permitted after consulting the promoter,
stated on the front page.
FACULTY OF BIOSCIENCE ENGINEERING
Interuniversity Programme
Master of Science in Food Technology (IUPFOOD)
Effect of structural modification of citrus and sugar beet
pectins on emulsifying capacity
Promoter: Prof. Dr. Ir. Marc Hendrickx Dissertation presented in fulfillment
Co-promoter: Dr. Ir. Stefanie Christiaens of the requirements for the degree of
Department for of Microbial and Molecular Systems Master of Science in Food Technology
Centre for Food and Microbial Technology
Gladys Kontoh
September 2015
i
ACKNOWLEDGEMENT
First and foremost, I would like to thank the Almighty God for his immerse grace, mercy and
protection throughout my research and Masters programme at large.
I would like to express my profound gratitude to my promoter, Prof. Dr. Ir. Marc Hendrickx
for his counsel, encouragement and guidance throughout the period of my research. In
addition I am thankful for the resources and time he invested into this research.
I would also like to thank my co-promoter, Dr. Ir. Stefanie Christiaens deeply for her
encouragement, advice and guidance during the research.
To my daily supervisor Clare Kyomugasho I express my heartfelt gratitude for her patience,
time, encouragement, advice and guidance throughout my research work. From her selfless
dedication to work, I profoundly learnt more on the need be dedicated in all that one carries
out in order to be very successful.
I am extremely grateful to all the staff of the Laboratory of Food Technology for the daily
support and encouragement. I also want to thank the Laboratory of Soft Matter, Rheology
and Technology (SMaRT) for the assistance with the viscosity analysis.
I would like to thank VLIR-UOS for the scholarship granted to me to pursue this Masters
programme.
Lastly, I want to thank my classmates, the Ghanaian students’ community in KU Leuven and
UGent, my family and friends for the love, support and encouragement throughout the
successful completion of my research and Masters programme.
ii
ABSTRACT
Pectin is a functional food ingredient extracted from the cell wall of several fruits and
vegetables and is used to improve the rheology of food products. Pectin, particularly from
sugar beet has been reported to possess emulsifying properties mainly due to its high
protein and acetylation content. However, due to its poor gelling ability, this pectin has not
been commercialized. On the other hand, the emulsifying potential of the commercialized
pectins such as citrus and apple pectin has not been extensively explored. This research
therefore is aimed at exploring the emulsifying potential of structurally modified commercial
citrus pectin in comparison to structurally modified sugar beet pectin.
To achieve this objective, commercial citrus pectin (CP) and sugar beet pectin (SBP) were
structurally modified by the action of carrot pectin methylesterase. Characterization of the
structure of the resulting pectin revealed that four levels of degree of methylesterification
(DM) of pectin including 16%, 35%, 65% and 95%, and two levels of DM (35% and 60%) were
obtained in CP and SBP, respectively. SBP exhibited a lower galacturonic acid content, higher
content of pectin-related neutral sugars, higher molar mass, higher amount of ferulic acid,
higher acetylation and protein content compared to CP. Exploring the flow behavior
revealed that the apparent viscosity of the pectin solutions increased with DM and pectin
concentration but decreased with pH. Furthermore, CP of low DM exhibited a shear thinning
behavior. Microscopy and particle size distribution studies revealed that CP of high DM and
SBP were better adsorbed onto the oil droplet surfaces compared to the low DM CP
samples. During storage of the emulsions, in general stability of both CP and SBP increased
with increasing pectin concentration and homogenization pressure but decreased with
increasing storage temperature, pH and DM of pectin. SBP emulsions were more stable than
CP for a given DM. For CP, low DM samples were more stable and probably stability of CP
was more dependent on the pectin concentration as well as the viscosity of solutions. For
pectin-protein emulsions explored in this study, the added protein did not improve stability.
In general, SBP samples portrayed better emulsifying and stabilizing capacity than citrus
pectin samples and this may be attributed to their structural differences.
iii
TABLE OF CONTENTS
ACKNOWLEDGEMENT ............................................................................................................................... i
ABSTRACT ................................................................................................................................................. ii
TABLE OF CONTENTS ............................................................................................................................... iii
LIST OF FIGURES ...................................................................................................................................... vi
LIST OF TABLES ...................................................................................................................................... viii
LIST OF ABBREVIATIONS AND SYMBOLS ................................................................................................. ix
GENERAL INTRODUCTION ....................................................................................................................... xi
6 GENERAL CONCLUSION ................................................................................................................. 78
REFERENCES .............................................................................................................................................. i
vi
LIST OF FIGURES
Figure 1.1: The primary structure of homogalacturonan methylesterified at C-6 and O-acetylated at
the O-2 or O-3. ........................................................................................................................................ 2
Figure 1.2: A schematic representation of the structural features of RG-I. (a) Linear galactan, (b)
arabinogalactan I, (c) branched arabinan and (d) hypothesized branched arabinan with galactan
Figure 5.18: (A) Emulsion stability study images of homogenized pectin-emulsions of citrus and sugar
beet pectin at different pHs after two weeks of storage at 4 °C and 35 °C. Homogenization pressure =
500 bar. ................................................................................................................................................. 75
viii
LIST OF TABLES
Table 5.1: A summary of the results of characterization of pectin structural elements. ..................... 43
Table 5.2: Apparent viscosity of citrus and sugar beet pectin solutions at a constant shear rate (5 s-1).
contents compared to citrus pectin (48.5 mg galactose /g pectin, 1.3 mg arabinose/g pectin and
8.8 mg rhamnose/g pectin). The significant amounts of rhamnose suggest a higher contribution
of RG-I in SBP. Furthermore, higher amounts of arabinose and galactose in SBP indicate more
“hairy” regions of pectin (Akhtar et al., 2002). Other neutral sugars including, xylose, mannose
and fucose were present in small amounts with fucose recording the least amount in both
pectin sources.
From the results of pectin characterization, it can be concluded that pectin from citrus was
more linear but exhibited a lower degree of acetylation, less protein and lower molar mass
46
compared to sugar beet pectin. After pectin characterization, the viscosity of watery pectin
solutions was determined and subsequently pectin-emulsions and pectin-protein emulsions
were prepared and examined for emulsifying properties.
5.2 Viscosity of pectin solutions
To gain insight into the rheological behavior of the pectin samples in solution, results obtained
for the flow behavior of the pectin solutions are discussed. The logarithm (log) of the viscosity
(Ns.m-2) was plotted as a function of time (s) in order to determine the apparent viscosity of the
pectin solutions at steady state. Results of the apparent viscosities of the pectin solutions are
shown in Table 5.2. More information on rheological behavior of the pectin solutions was
established by plotting the log of apparent viscosities (Ns.m-2) against shear rate (s-1) (Figures
5.2 and 5.3).
5.2.1 Effect of DM and pH on the viscosity of pectin solutions
Results obtained at constant shear rate (5 s-1) exhibited a decrease in apparent viscosity as the
DM of pectin increased at any given pH and pectin concentration as shown in Table 5.2. Both CP
and SBP showed similar trends. For instance, in citrus pectin solutions (0.5% w/v) at pH 3.0, the
apparent viscosity values decreased from 93.6 x 10-4 Ns.m-2 for CP 16 to 23.3 x 10-4 Ns.m-2 for CP
95. SBP samples also showed a decrease in apparent viscosity from 77.3 x 10-4 Ns.m-2 for SBP 35
to 32.6 x 10-4 Ns.m-2 for SBP 60.
When the pH was increased to 4.2 and 6.2, the viscosity of both CP and SBP samples decreased
except for CP 95 which was more or else constant with increasing pH. As pH increases, repulsion
increases, therefore limiting pectin interactions within the aqueous solution. For the lower DMs,
as pH increases more COO‾ groups are charged leading to more repulsion thus less chances of
entanglement to create a dense network. It was also suggested by Fraeye et al. (2010) that as
pH decreases protonation of carboxyl groups is accompanied by a transition of pectin from a
two-fold to a three-fold conformation with the three-fold helices probably cross linking mainly
by hydrogen bonds. Such complexation may have been strong enough in the lower DMs thus
47
giving some resistance to unfolding hence the higher viscosity. On the other hand for CP 95,
irrespective of pH increase the number of chargeable groups is too low therefore no build up in
repulsion is attained leading to a constant viscosity (Axelos et al., 1991).
Table 5.2: Apparent viscosity of citrus and sugar beet pectin solutions at a constant shear rate (5 s-1).
pH 3.0
DM Viscosity (Ns m-2) @ 5 s-1shear rate
0.1% 0.5% 1%
CP 16 18.6 x 10-4 93.6 x 10-4 632 x 10-4
CP 35 15.4 x 10-4 52.1 x 10-4 206 x 10-4
CP 65 15.5 x 10-4 40.5 x 10-4 163 x 10-4
CP 95 15.2 x 10-4 23.3 x 10-4 38.8 x 10-4
SBP 35 18.6 x 10-4 77.3 x 10-4 445 x 10-4
SBP 60 14.8 x 10-4 32.6 x 10-4 107 x 10-4
pH 4.2
CP 16 16.7 x 10-4 59.0 x 10-4 284 x 10-4
CP 35 18.0 x 10-4 39.9 x 10-4 178 x 10-4
CP 65 15.3 x 10-4 41.4 x 10-4 158 x 10-4
CP 95 13.9 x 10-4 24.6 x 10-4 48.1 x 10-4
SBP 35 18.0 x 10-4 53.9 x 10-4 224 x 10-4
SBP 60 20.0 x 10-4 37.2 x 10-4 116 x 10-4
pH 6.2
CP 16 14.0 x 10-4 26.6 x 10-4 105 x 10-4
CP 35 14.2 x 10-4 19.6 x 10-4 100 x 10-4
CP 65 15.2 x 10-4 26.4 x 10-4 96.5 x 10-4
CP 95 15.3 x 10-4 27.0 x 10-4 45.1 x 10-4
SBP 35 19.8 x 10-4 43.0 x 10-4 142.0 x 10-4
SBP 60 17.5 x 10-4 40.1 x 10-4 111.0 x 10-4
In general, the LM pectins exhibited higher apparent viscosities than HM pectins of each pectin
source (Table 5.2).
Comparing the viscosities of citrus and sugar beet pectin solutions with a similar DM (SBP 35
and CP 35), SBP 35 had higher viscosity than CP 35. For instance CP 35 and SBP 35 solutions
(0.5%w/v) at pH 3.0 had apparent viscosities of 52.1 x 10-4 Ns m-2 and 77.3 x 10-4 Ns m-2,
respectively. This may be due to the higher acetyl content in sugar beet pectin than in citrus
pectin (cf. Table 5.1) where the acetyl groups may have increased the solubility of pectin in the
48
aqueous medium by lowering the hydrophobicity of the pectin (Gou et al., 2012). This improves
pectin-solvent interactions as well as increases the ability of pectin to modify the viscosity of the
aqueous medium (Surh et al., 2006). In addition, the presence of more neutral sugars in SBP
compared to CP may have also improved SBP’s hydrophilic nature owing to the hydroxyl groups
of neutral sugars side chains protruding into the aqueous medium (Akhtar et al., 2002).
5.2.2 Effect of pectin concentration on the viscosity of pectin solutions
At a given pH, the apparent viscosity of pectin solutions increased significantly with increasing
pectin concentration (Table 5.2). This is in agreement with the findings of Einhorn-Stoll et al.
(2005) where increasing pectin concentration increased viscosity irrespective of the DM. In
addition, as solutions of high pectin concentrations are more concentrated than those of lower
pectin concentrations, the pectin molecules in the former tend to gradually come into contact
with one another until their movement is restricted leading to an enhancement in viscosity of
the solution (Milani and Maleki, 2012).
5.2.3 Effect of DM and pH on the viscous behavior of pectin solutions at varying shear rates
Examining the behavior of pectin under varying shear rates showed that increasing shear rate at
a given pH had an influence on the viscous behavior of different DMs of the various pectin
solutions. Looking at the trends of 1% citrus pectin concentrations at pH 3.0, the viscosities of
CP 16 and CP 35 decreased with increasing shear rate (Figure 5.2). On the other hand, the
viscosities of CP 65 and CP 95 were only slightly influenced by increasing shear rate. Similarly,
the sugar beet pectin samples, SBP 35 and SBP 60, both exhibited slight influence with
increasing shear rate. Similar trends were observed at both pH 4.2 and pH 6.2 (Figure 5.2) and
the influence of shearing increased with increasing pH especially for pH 6.2. The decrease in
may be due to the loss in the network of the entanglements present in the LM citrus pectin
samples (CP 16 and CP 35) due to the increase in the applied force (Surh et al., 2006).
Furthermore, owing to the fact that the molecules of the more viscous solutions orient
perpendicular to the surface of the spindle surface, faster rotation destroys the structure of the
49
solution. Thus, LM pectins depict more shear thinning flow properties than the HM pectin
solutions (Rao, 2013; Saha and Bhattacharya, 2010) structure of the solution.
Figure 5.2: Viscosity (log) of the pectin solutions at different pHs at varying shear rates (log).
5.2.4 Impact of pectin concentration on the viscous behavior of pectin solutions at varying
shear rates
At any given pH (for example pH 3.0) increasing the pectin concentration from 0.1% to 1%,
resulted in more shear thinning behavior of the pectin solutions (Figure 5.3). Similar
observations were made at pH 4.2 and pH 6.2 except that a more shear thinning behavior was
observed as the pH increased. The lower pectin concentrations (dilute solutions) may probably
have formed very few entanglements (if any) that freely dispersed in the solution. As a result,
increasing the stress on the sample did not bring a significant decrease in viscosity (Milani and
Maleki, 2012). On the other hand, for the higher pectin concentrations owing to the several
entanglements formed, increasing stress significantly decreases their viscosity as their stiff
entanglements were gradually destroyed (Milani and Maleki, 2012; Rao, 2013).
50
Figure 5.3: Viscosity (log) of solutions with different pectin concentrations at varying shear rates (log).
In conclusion, the LM citrus pectin solutions may be viewed as pseudo-non Newtonian fluids at
higher pectin concentrations owing to their shear thinning behavior i.e. decreasing viscosity as
shear rate. In addition, at a lower shear rate such fluids are more viscous compared to at a
higher shear rate (Marcotte et al., 2001; Rao, 2013). On the other hand, at the lowest pectin
concentration, pectin solutions from both soures could be viewed as Newtonian (Saha and
Bhattacharya, 2010).
5.3 Microscopy and particle size distribution of the ultra-turaxed emulsions
Microscopy examination was carried out to investigate the emulsifying capacity of the pectin,
i.e. degree of adsorption of the pectin to the oil droplet surface. To facilitate this, different pHs
were used; pH 3.0 slightly below pKa value of pectin (3.5-4.10) (Kyomugasho et al., 2015b), pH
4.2 which is approximately the pKa value of pectin and pH 6.2 which is above pKa value of
pectin. Additionally, pH 3.0 and 4.2 are below the pI of protein (specifically pH 4.7 for BSA)
(Peng et al., 2005) while pH 6.2 is above the pI value of protein. As such, interactions between
partially positive protein and negatively charged pectin would be expected around pH 4.2
51
whereas electrostatic repulsion between pectin and protein would be expected at pH 6.2 (Surh
et al., 2006).
Results of particle size distribution (PSD) of the emulsions at pH 4.2 and 6.2 as well as results of
fluorescence microscopy examinations are presented here.
5.3.1 Adsorption of pectin onto the oil droplet surface (emulsifying potential of pectin)
This was evaluated by ultra-turaxing watery pectin solutions in presence of olive oil followed by
microscopy examination of the emulsions as well as measurement of the PSD. Ultra-turaxed
samples were preferred as droplet sizes were easily viewed under the microscopy. In the case of
homogenized samples, the droplets were too small to facilitate this examination. However, after
storage microscopy examination of these samples was carried out.
5.3.1.1 Effect of DM and pH on adsorption of pectin onto the oil droplet surface (O/W emulsions)
From Figure 5.4 it can be observed that in citrus pectin emulsions at pH 4.2, the droplet sizes
decreased with increasing DM. In this case, at 0.5% pectin, CP 16 exhibited the largest droplet
size while CP 95 depicted the least droplet size. This was in agreement with studies by Verrijssen
et al. (2014) with HM pectin showing smaller droplet sizes compared to LM pectin. It is
suggested that the presence of more methyl groups in HM pectin partake in hydrophobic
interactions that sufficiently reduce the interfacial tension between the oil/water interface
which improves the emulsifying potential of HM pectin hence the smaller particle sizes (Van
Buren, 1991). On the other hand, for LM pectin presence of few methylester groups limited
hydrophobic interactions thus the larger droplets observed.
When the pH of the solutions was increased to 6.2, a similar trend of droplet size increasing
with decreasing DM was observed but with the droplets at this pH being slightly bigger than at
pH 4.2. In the case of CP 16 this was more pronounced. A possible explanation may be that at
pH 6.2 which is above the pKa of pectin, charge repulsion between the pectin molecules
coupled with the limited hydrophobic interactions (due to few methylester groups) may have
52
reduced the ability of pectin to approach the oil droplets giving rise to larger droplet sizes
(Aronson and Petko, 1993; Verheul and Roefs, 2004).
In the case of SBP, similar observations were made, with smaller sizes observed for SBP 60
compared to SBP 35 which can be attributed to methyl ester groups. Furthermore, comparing
CP and SBP of approximately similar DMs (SBP 35 and CP 35 in Figure 5.4) slightly smaller
droplets were seen for SBP at pH 6.2. Perhaps this can be attributed to the higher protein and
acetyl content of sugar beet pectin. The acetyl groups provide a level of steric hindrance while
the protein ably reduces the interfacial surface tension of the oil droplets facilitating effective
adsorption of the pectin (Dickinson, 2010; Ralet et al., 2003; Surh et al., 2006) thus allowing
formation of smaller droplets in SBP 35. The higher molar mass and and neutral sugars may
have facilited the greater adsorption in SBP. In addition, studies by Siew and Williams, (2008)
suggested that fractions of the adsorbed pectin are rich in ferulic acid. As such, SBP’s higher
ferulic acid content may have also enhanced its adsorption onto the oil droplet surfaces.
To gain more insight into the results observed in the microscopy examination, particle size
distribution was measured as mentioned earlier. From PSD results, it can be observed that
bimodal (two peaks) and multimodal (more than two peaks) distributions were obtained.
According to Trotta et al. (2001) this is expected if only a centripetal force such as ultra-turaxing
is applied. This results in non-homogenously distributed droplets in emulsions. PSD plots of CP
16 to CP 95 confirmed the observation of microscopy examination, with peak maxima shifting
more to the left with increasing DM indicating lower particle size as shown in Figure 5.5A and
5.5B. A similar trend was seen for the emulsions at pH 6.2 (Figure 5.5B).
53
pH 4.2 (0.5%) pH 6.2 (0.5%)
CP 16
500 µm
CP 35
500 µm
500 µm 500 µm
500 µm
CP 65
500 µm
500 µm
500 µm 500 µm
CP 95
SBP 35
SBP 60
pH 4.2 (0.5%) pH 6.2 (0.5%)
500 µm 500 µm
500 µm500 µm
Figure 5.4: Fluorescence microscopy images of citrus and sugar beet pectin emulsions at pH 4.2 and pH 6.2. Scale bar = 500 µm.
54
B A
Figure 5.5: PSD of citrus and sugar beet pectin emulsions of 0.5% pectin at (A) pH 4.2 and (B) pH 6.2.
Generally, comparing citrus and sugar beet pectin emulsions of a similar DM, it can be observed
that the PSD of CP 35 was larger than that of SBP 35 (Figure 5.5A and 5.5B). The higher amount
of acetyl groups and protein content in sugar beet as compared to citrus pectin may have
accounted for the differences since smaller droplet sizes suggests better emulsifying capacity of
pectin. The explanation for the results of PSD is similar to that of microscopy results.
5.3.1.2 Effect of pectin concentration on adsorption of pectin onto the oil droplet surface
In citrus pectin, increasing the pectin concentration resulted in an increase in droplet size. For
instance in citrus pectin emulsions of CP 16 at pH 4.2, the oil droplet sizes increased with
increasing pectin concentration as shown in Figure 5.6. Results of PSD confirmed this result with
the curve of 1% pectin concentration shifting more to the right compared to emulsions with
55
0.1% pectin 1% pectin
CP 16
500 µm
pH 4.2
CP 35
CP 65
CP 95
SBP 35
SBP 60
500 µm 500 µm
500 µm 500 µm
500 µm 500 µm 500 µm
500 µm 500 µm
500 µm 500 µm
500 µm 500 µm
Figure 5.6: Fluorescence microscopy images of citrus and sugar beet pectin emulsions at different pectin
concentrations. Scale bar = 500 µm.
56
A B
Figure 5.7: PSD plots of citrus and sugar beet pectin emulsions at pH 4.2 with (A) 0.1 % and (B) 1% pectin
concentrations.
0.1% pectin, indicating larger average particle sizes for 1% concentration as shown in Figure
5.7A and 5.7B. A similar trend was observed for CP 35 and CP 65 except CP 95 which decreased
in droplet size with increasing pectin concentration (Figure 5.7A and 5.7B). At a higher pectin
concentration, probably more repulsion of the pectin molecules occurs thus larger oil droplets
formed. With increasing pectin concentration, aggregate-like structures were observed at pH
4.2 in the rest of the DMs except DM 95. This may be attributed to the low electrostatic
repulsion forces between the pectin and oil droplets causing a decrease in the steric effect.
Therefore, several droplets may have come together through interactions of the polymer at
their surfaces leading to bridging flocculation hence the aggregate-like structures formed
(Doublier et al., 2000; Guzey and McClements, 2007). In addition, as suggested by Gharsallaoui
et al. (2010), at very high pectin concentration the surface of more than one droplet may be
adsorbed by the pectin molecules causing bridging flocculation. It is also possible that at a high
57
concentration, pectin forms a thick macromolecular multilayer (Dickinson and James, 2000;
Huang et al., 2001) thus the larger particle sizes.
In addition, the sugar beet pectin samples also showed an increase in droplet size with
increasing pectin concentration at pH 4.2 (Figure 5.6). Oil droplet aggregation at pH 4.2
especially in the 1% pectin emulsions of both SBP 35 and SBP 60 was probably due to the
concetraction effect causing bridging flocculation and low repulsion coupled with pectin-protein
interactions. At this pH protein is positively charged and pectin slightly negatively charged and
therefore their interaction may have enhanced the likelihood of aggregation occurring. On the
other hand, for both SBP 35 and SBP 60 at pH 6.2, no droplet aggregation was observed when
the pectin concentration was increased from 0.1 to 1%. This may be due to repulsion between
the negatively charged pectin molecules or between pectin and protein molcules at this pH
(Williams et al., 2004).
The PSD plots also showed differences in the patterns of the different pectin concentrations at
pH 4.2 (Figure 5.7). The PSD curves of the SBP emulsions shifted to the right (showing increased
particle sizes) as the concentration of pectin in the emulsions increased from 0.1% to 1%. In
addition, SBP 35 emulsions with 1% pectin depicted a multimodal distribution compared to
bimodal distribution by SBP 60 (Figure 5.7B). This was probably due to aggregate formation as
seen from the microscopy images where SBP 35 showed more aggregation than SBP 60 (Figure
5.6).
In conclusion, at a particular pH and pectin concentration the droplet sizes of both CP and SBP
pectin emulsions increased with decreasing DM. Increasing the pH to a pH above the pKa of
pectin and pI of protein led to an increase in the droplet sizes of the lower DMs. Furthermore,
the droplet sizes of the emulsions increased with increasing pectin concentration. Finally, sugar
beet pectin emulsions exhibited smaller particle sizes compared to citrus pectin samples of a
similar DM. It can be observed that CP samples of high DM and SBP samples were better
adsorbed on to the oil droplet surfaces than low DM pectin samples of CP.
58
5.4 Emulsion stability study
For this section, emulsions were prepared by ultra-high pressure homogenization.
Homogenization of the emulsions was aimed at producing emulsions of smaller droplet sizes for
greater stability (Williams et al., 2004). The homogenized emulsions containing pectin of
different DMs were analyzed for particle size distribution (PSD) before and during storage. For
end of storage results, images of emulsions as well as results of PSD at the highest storage
temperature (35 °C) will be shown as the changes in emulsion stability (if any) would be more
prominent at this temperature. This is based on the hypothesis that reactions in a food system
increase when the kinetic energy of the system increases leading to an increase in the thermal
energy of oil droplets. As such, droplet collisions increases due to loss of electrostatic repulsion
which renders the system unstable (Freitas and Müller, 1998). For interpretation of the PSD
plots, denotations d10, d50, d90 and D [4,3] are used, with d10, d50 and d90 representing the
particle diameter that 10%, 50% and 90% of the particles in the emulsion are smaller than,
respectively. In this research d90 of the PSD, will be explored. In addition, the pattern of PSD
measurements depicted will be described as monomodal, bimodal or multimodal depending on
the number of peaks either one, two or more peaks, respectively.
Immediately after homogenization all samples were physically similar and therefore to establish
differences or similarities in the emulsions, PSD was used.
After storage at temperatures of 4 °C, 20 °C and 35 °C for up to eight weeks, the effect of
several properties including pH, pectin concentration, applied homogenization pressure, added
protein, storage time and storage temperature on emulsion stability were examined. The results
obtained are presented and discussed below. Furthermore, the possible destabilization
mechanisms occuring during storage were investigated through microscopy examinations of the
emulsions.
59
5.4.1 Effect of DM and pH on stability of ultra-high pressure homogenized emulsions before
and after storage
A) Pectin emulsions
Before storage, for example, in citrus pectin emulsions of 0.5% pectin concentrations at pH 3.0
(Figure 5.8), it can be seen that the PSD of CP 16, CP 35 and CP 65 emulsions depicted similar
monomodal distributions of almost the same average particle size with slight differences in
%volume fraction. On the other hand, the PSD of CP 95 emulsions shifted more to the right
suggesting larger average particle diameter compared to the other citrus pectin emulsions. This
may be attributed to the very low amount of carboxylic groups in CP 95 thus no pectin
interactions occur (Axelos et al., 1991). Owing to this, the non-adsorbed oil droplets may have
aggregated or coalesced leading to final droplets of large sizes. (Axelos et al., 1991; Rao, 2013).
In the case of SBP, pectin emulsion of both SBP 35 and SBP 60 displayed similar PSD patterns of
monomodal distribution at pH 3.0 as shown in Figure 5.8.
When the pH was increased to 4.2 and 6.2, in citrus pectin-emulsions it could be observed that
as the DM increased from CP 16 to CP 95 PSD curves changed from monomodal to a multimodal
distribution with CP 16 shifting towards the right (large average droplet sizes) while CP 95
shifted to the left (towards smaller droplet sizes) as shown in Figure 5.8. In CP 95, perhaps due
to the low carboxylic groups that were charged, increasing the pH may not have led to any build
up in repulsion hence pectin molecules approach each other leading to the observed smaller
droplet sizes (Axelos et al., 1991). Furthermore, probably the high methyl groups in CP 95 may
have sufficiently decreased the interfacial tension through hydrophobic interactions resulting in
smaller average droplet sizes (Van Buren, 1991; Verrijssen et al., 2014).
60
Before storage
Figure 5.8:Particle size distribution curves of citrus and sugar beet pectin-emulsions at different pHs
before storage. Homogenization pressure = 250 bar.
After two weeks of storage, it can be observed from Figure 5.9A that for CP, stability was higher
in the low DM samples. Furthermore, the stability images showed a decrease in emulsion
stability with increasing pH (Figure 5.9A). For instance at pH 3.0, CP 16, CP 35, were still slightly
stable while CP 65 and CP 95 were visibly less stable. These changes became more pronounced
as pH increased (more clear phase separation) (Figure 5.9A). From the microscopy examination,
phase separation at pH 6.2 was mainly due to aggregation whereas coalescence was observed
at pH 4.2. As suggested by Gancz et al. (2005) and Gharsallaoui et al. (2010), at higher pH (above
the pKa of pectin) there is charge repulsion between the pectin molecules leading to larger
droplet sizes because of the limited compactness between the pectin molecules. This may have
led to greater degree of separation at the higher pH. On the other hand, in the case of SBP, both
emulsions appeared to exhibit less phase separation compared to the citrus pectin-emulsions
(Figure 5.9A). Sugar beet pectin’s higher protein and ferulic acid contents (cf. Table 5.1) than
citrus pectin may have accounted for its great emulsifying as well as stabilizing effect as
suggested by Leroux et al. (2003) and Siew and Williams, (2008). Furthermore, it is reported that
highly branched sugar beet pectin improves long term emulsion stability by covering the oil
61
effectively as well as the hydroxyl groups of the side chains improving the hydrophilic nature of
the pectin (Akhtar et al., 2002; Jung and Wicker, 2012).
2 W at 35 °C
35 ° C
pH 3.0
pH 4.2
pH 6.2
CP 16 CP 35 CP 65 CP 95 SBP 35 SBP 60
Figure 5.9: (A) Emulsion stability study images of homogenized pectin emulsions (0.5% pectin) at
different pHs after two weeks of storage at 35 °C. Homogenization pressure = 250 bar.
62
0
2
4
6
8
0.01 0.1 1 10 100 1000
%V
olu
me
Particle diameter (µm)
0.5% pectin - pH 3.0
0
2
4
6
8
0.01 0.1 1 10 100 1000
%V
olu
me
Particle diameter (µm)
0.5% pectin - pH 6.2
0
2
4
6
8
0.01 0.1 1 10 100 1000
%V
olu
me
Particle diameter (µm)
0.5% pectin - pH 4.2
After storage
Figure 5.9: (B) Particle size distribution patterns of citrus and sugar beet pectin emulsions at different
pHs after two weeks of storage. Homogenization pressure = 250 bar.
As the pH increased to 4.2 and 6.2, all the partcile size in CP emulsions shifted towards larger
average particle sizes. A similar trend of increasing average particle size as pH increased was
observed in the sugar beet pectin samples (Figure 5.9B). Loss of electrostatic repulsions and/or
steric hindrance may have occurred during the storage period leading to loss of emulsion
stability (Guzey and McClements, 2007; Lopez-Sanchez and Farr, 2012)
B) Pectin-protein emulsions
Before storage, for pectin-protein emulsions (pectin emulsions with added BSA protein),
generally for CP pectin-protein emulsions, an increase in average particle size was observed
compared to pectin emulsions of CP (results not shown). As the DM increased from CP 16 to CP
95, monomodal and bimodal PSD was observed (Figure 5.10). At pH 3.0, the PSD of the CP 16
pectin-protein emulsion shifted to the right indicating larger particles while CP 95 shifted more
to the left indicating the least average particle diameter (Figure 5.10). Pectin around its pKa has
about 50% chargeable COO‾ groups which decrease as pH decreases hence being weakly
charged. For CP 95 probably the methyl groups are responsible for smaller size. As such, CP 16
63
with few methyl groups showed larger sizes. For low DMs, considering that at this pH, ionisation
of carboxylic groups is limited repulsions is less, therefore molecules can approach each other
may have led to pectin-protein interactions through associative ionic bonding of the few weakly
charged carboxylic groups (Lopes da Silva et al., 1994). This may have led to aggregation or
coalescence of droplets resulting in larger measured droplet sizes (Axelos et al., 1991; Dickinson,
2003). On the other hand, despite the presence of added protein in CP 95 emulsions, no
influence of protein was observed because of the very low chargeable carboxylic groups present
in CP 95 which did not seem to effectively initiate pectin-protein interactions (Axelos et al.,
1991; Dickinson, 1998; Kar and Arslan, 1999).
In the case of pectin-protein emulsions at pH of 3.0 prepared with SBP, SBP 60 shifted more to
the left depicting the smaller particle sizes compared to SBP 35 (Figure 5.10). But as the pH
increased to 4.2, SBP 35 shifted to the left with significant decrease in %volume fraction
compared to SBP 60 as shown in Figure 5.10. This could be explained by interactions between
the protein and the high number of carboxylic groups in the LM pectin (SBP 35) than in the HM
0
2
4
6
8
10
0.1 1 10 100 1000
%V
olu
me
Particle diameter (µm)
0.1 % - pH 3.0
0
2
4
6
8
10
0.1 1 10 100 1000
%V
olu
me
Particle diameter (µm)
0.1% - pH 4.2
0
2
4
6
8
10
0.1 1 10 100 1000
%V
olu
me
Particle diameter (µm)
0.1% - pH 6.2
Before storage
Figure 5.10: Particle size distribution curves of citrus and sugar beet pectin-protein emulsions at different
pHs before storage. Homogenization pressure = 250 bar.
64
pectin (SBP 60) there by forming a thick protective layer around the oil droplets which led to
larger droplet sizes (Akhtar et al., 2002). As the pH was further increased to 6.2, both samples
exhibited similar PSD patterns implying possible negligible influence of DM at this pH. In
addition, probably the carboxylic groups of SBP 60 also got fully charged at pH 6.2, which is
above the pKa of pectin. Strong repulsive forces of almost equal magnitude between strongly
negative pectin and negatively charged protein within both samples at this pH may account for
the similar PSD patterns observed (Surh et al., 2006).
In the case of pectin-protein emulsions in relation to the influence of pH, after two weeks of
storage at pH 3.0, although the addition of protein resulted in some degree of phase separation
in the CP 16 pectin-protein emulsion compared to the CP 16 pectin emulsions (Figure 5.11A), a
‘slight’ improvement in stability was observed in CP 65 and CP 95 pectin-protein emulsions
compared to their pectin-emulsions (Figures 5.11A). These observations were supported by the
PSD plots, where CP 16 demonstrated larger particles compared to the CP 65 and CP 95 (Figure
5.11B).
At low pH such as pH 3.0, HM pectins are more susceptible to hydrogen bonds and hydrophobic
interactions (example with protein) between the methyl ester groups which leads to
stabilization of the food system. On the other hand, LM pectins form strong electrostatic bonds
through cross linking with increasing ionic strength and pH (Thakur et al., 1997; Williams, 2011).
Furthermore, when the pH was increased from 4.2 to pH 6.2, for the low DMs of both CP and
SBP a slight “improvement” in stability could be proposed. Interestingly, at pH 6.2 all the citrus
pectin-protein emulsions appeared similar (from the stability images) (Figure 5.11A). This was
supported by the PSD plots which showed similar monomodal distribution patterns as well as
similar average sizes compared to bimodal and multimodal distributions of their pectin-
emulsions as shown in Figure 5.11B. Probably pectin-protein emulsions had all fully destabilized
hence the similar poor stabilities observed.
65
35 °C pectin emulsions (0.1%)
pH 4.2 pH 6.2 pH 4.2 pH 6.2
CP 16
CP 65
CP 95
SBP 35
SBP 60
35 °C pectin-protein emulsions (0.1%)
pH 3.0pH 3.0
Figure 5.11:(A) Emulsion stability images of homogenized pectin and pectin-protein emulsions at
different pHs after two weeks of storage at 35 °C. Homogenization pressure = 250 bar.
66
0
2
4
6
8
10
0.1 1 10 100 1000
%V
olu
me
Particle diameter (µm)
0.1% - pH 3.0 (pectin-protein emulsions)
0
2
4
6
8
10
0.1 1 10 100 1000
%V
olu
me
Particle diameter (µm)
0.1% - pH 4.2 (pectin-protein emulsions)
0
2
4
6
8
10
0.1 1 10 100 1000
%V
olu
me
Particle diameter (µm)
0.1% - pH 6.2 (pectin-protein emulsions)
After storage
Figure 5.11: (B) Particle size distribution curves of citrus and sugar beet pectin and pectin-protein
emulsions at different pHs after two weeks of storage. Homogenization pressure = 250 bar.
Regarding sugar beet pectin-protein emulsions, the stability of SBP35 was lowest at pH 3.0 but
as pH increased, less phase seperation was observed. At low pH, possibly the high number of
methyl groups of SBP 60 ensured less phase seperation than in the case of SBP 35. Less
creaming is observed in pectin-protein emulsions compared to pectin-emulsions of SBP. At pH
4.2, the interaction between pectin and protein probably provides some degree of stability
while at pH 6.2 stability can be attributed to repulsion forces.
5.4.2 Effect of pectin concentration on stability of ultra-high pressure homogenized
emulsion before and after storage
A) Pectin emulsions
Before storage, at a given homogenization pressure and pH, for CP emulsions average particle
sizes decreased with increasing pectin concentration for high DMs while for the low DM and a
slight increase in particle size may be suggested. Looking at Figure 5.12, CP 16 at 250 bar
attained a monomodal distribution with increasing pectin concentration from 0.1% to 1%. In
addition, a slight shift of PSD of droplets to the right was observed, indicating increasing droplet
67
size. CP 35 emulsions showed similar trends (Figure 5.12). On the other hand, the PSD of CP 65
and CP 95 shifted more to the left showing lower particle sizes.
For the sugar beet pectin emulsions, as the pectin concentration increased from 0.1% to 1%,
SBP 60 attained a monomodal distribution suggesting more uniformly dispersed droplets in the
emulsions as shown in Figure 5.12. SBP 35 remained monomodal in particle distribution with
increasing pectin concentration and increasing %volume fraction. Comparing the sugar beet
pectin samples to the citrus pectin samples, PSD of SBP did show prominent shifts with
increasing pectin concentration (Figure 5.12).
0
2
4
6
8
10
0.01 0.1 1 10 100 1000
%V
olu
me
Particle diameter (µm)
0.1% pectin - pH 4.2
0
2
4
6
8
10
0.01 0.1 1 10 100 1000
%V
olu
me
Particle diameter (µm)
0.5% pectin - pH 4.2
0
2
4
6
8
10
0.01 0.1 1 10 100 1000
%V
olu
me
Particle diameter (µm)
1% pectin - pH 4.2
Before storage
Figure 5.12: Particle size distribution curves of citrus and sugar beet pectin emulsions at different pectin
concentrations before storage. Homogenization pressure = 500 bar.
After two weeks of storage, it was observed that increasing the pectin concentration of pectin
emulsions from 0.1% to 1% showed increase in the stability of the emulsions irrespective of the
pH and DM. As can be seen from images in Figure 5.13A, pectin-emulsions at 0.1% showed a
greater degree of separation compared to those at 1%. The phase separation of 0.1% emulsions
was as a result of oil droplet growth due to coalescence as seen from the microscopy
68
examinations after storage (results not shown). For CP separation became more prominent with
increasing DM and emulsions of SBP were still more stable at 0.5% (Figure 5.13A). From Figure
5.13B, the PSD plots showed that the different pectin concentrations of the pectin emulsions
exhibited bimodal and multimodal distributions. Nonetheless, the 1% pectin concentration
emulsions showed the best stabilization (Figure 5.13A). Probably in these emulsions sufficient
covering of the oil droplets enhances emulsion stability, higher pectin concentration is required
for such a phenomenon (Leroux et al., 2003). This supports the observation of increased
2 W at pH 4.2
35 ° C
0.1%
0.5%
1%
CP 16 CP 35 CP 65 CP 95 SBP 35 SBP 60
Figure 5.13: (A) Emulsion stability study images of homogenized pectin emulsions at different pectin
concentrations after two weeks of storage at 35 °C. Homogenization pressure = 500 bar.
69
After storage
Figure 5.13: (B) Particle size distribution curves of citrus and sugar beet pectin emulsions at different
pectin concentrations after storage. Homogenization pressure = 500 bar.
stability as concentration of pectin increased. It is likely that the higher viscosities depicted by
high pectin concentration solutions gave rise to stable matrices which resisted environmental
conditions and might have enhanced the stability (Saha and Bhattacharya, 2010). This was
affirmed from the viscosity results of the pectin solutions where apparent viscosity increased
with increasing pectin concentration at a given DM and pH as shown in Figure 5.3 and Table 5.2.
In general, it can be observed that the 0.1% emulsions were relatively unstable at the different
conditions.
B) Pectin-protein emulsions
Regarding the pectin-protein emulsions before storage, the PSD of CP 16 pectin-protein
emulsions shifted more to the right showing an increase in particle size compared to CP 16
pectin emulsions (results not shown). The presence of protein may have enhanced pectin-
protein interactions especially in the LM pectin, CP 16, leading to thick protective layers of
protein and pectin around the droplets which resulted in overall droplets of larger sizes (Guzey
and McClements, 2007) or even aggregation. On the other hand, PSD of CP 65 and CP 95 pectin-
70
protein emulsions shifted more to the left compared to their pectin emulsions even after
storage as shown in Figure 5.14. This maybe attributed to the low amount of carboxylic groups
in the HM pectins resulting in limited pectin-protein interactions especially in CP 65 whereas in
CP 95 probably no interactions may have occurred (Axelos et al., 1991; Gancz et al., 2005; Rao,
2013). Similarly, for the sugar beet pectin-protein emulsions, SBP 35 shifted more to the right
compared to its pectin emulsions, while SBP 60 shifted more to the left towards the smaller
particle sizes compared to larger particle sizes of its pectin emulsions (even after storage as
shown in Figure 5.14). Owing to the high amount of carboxylic groups present in SBP 35, pectin-
protein interactions were enhanced and may have led to a thick layer around the oil droplets
hence the larger sizes measured (Siew and Williams, 2008) or aggregation of droplets may have
cause the larger observed size.
0
2
4
6
8
10
0.1 1 10 100 1000
%V
olu
me
Particle diameter (µm)
0.1 % - pH 3.0 (pectin-protein emulsions)
0
2
4
6
8
10
0.1 1 10 100 1000
%V
olu
me
Particle diameter (µm)
0.1% - pH 3.0 (pectin-protein emulsions)
After storageBefore storage
Figure 5.14: Particle size distribution plots of pectin-protein emulsions of citrus and sugar beet pectin of
0.1% pectin and 0.1% protein concentrations at pH of 3.0 before and after storage. Homogenization
pressure = 500 bar.
After storage, the pectin-protein emulsions generally depicted an increase in droplet size at a
given pectin concentration of 0.1% (Figure 5.14).
71
5.4.3 Effect of pressure on stability of ultra-high pressure homogenized emulsions before
and after storage
A) Pectin emulsions
Before storage, the impact of pressure on PSD of the CP emulsions of different DMs showed a
shift of PSD towards the smaller particle sizes (to the left) as pressure increased (Figure 5.15). As
the homogenization pressure increased from 250 to 1000 bar, the sugar beet pectin emulsions
also showed similar trends with average particle sizes decreasing with increasing pressure
(Figure 5.15). The lower pressures applied probably resulted in final droplet sizes of larger sizes
due to the time for droplet-droplet coalescence or aggregation being shorter than the sufficient
time required for adsorption of the pectin while at higher applied pressure, rapid stabilization of
the droplets against coalescence was attained (Surh et al., 2006; Williams et al., 2004). For the
sugar beet pectin emulsions a slight increase in average droplet size was noticed with
decreasing applied pressure. The higher ferulic acid and protein content in sugar beet pectin
compared to citrus pectin may have enhanced SBP adsorption onto the droplet surface (Akhtar
et al., 2002; Siew and Williams, 2008; Yuan et al., 2008).
0
2
4
6
8
10
0.01 0.1 1 10 100 1000
% V
olu
me
Particle diameter (µm)
0.5% pectin - pH 4.2
250 bar
0
2
4
6
8
10
0.01 0.1 1 10 100 1000
% V
olu
me
Particle diameter (µm)
0.5% pectin - pH 4.2
500 bar
0
2
4
6
8
10
0.01 0.1 1 10 100 1000
% V
olu
me
Particle diameter (µm)
0.5% pectin - pH 4.2
1000 bar
Before storage
Figure 5.15: Particle size distribution plots of the pectin emulsions of citrus and sugar beet pectin at
different homogenization pressures for a given pH and pectin concentration before storage.
72
CP 16
0.5%
CP 35 CP 65 CP 95 SBP 35 SBP 602 weeks pH 4.2
at 35 °C250 500 1000 bar 250 500 1000 bar 250 500 1000 bar 250 500 1000 bar 250 500 1000 bar 250 500 1000 bar
Figure 5.16: (A) Emulsion stability study images (after two weeks) of pectin emulsions homogenized at different pressures and stored at 35 °C.
After storage
Figure 5.16 (B): Particle size distribution patterns of citrus and sugar beet pectin emulsions at different homogenization pressures after storage.
73
Regarding the pectin emulsions stability after storage, the stability of pectin-emulsions
increased with increasing pressure as shown in Figure 5.16A after two weeks of storage.
Emulsions at 250 bar exhibited a higher degree of separation compared to those at 1000 bar
(especially in the low DM CP emulsions). For the 1% emulsions, microscopy examination
revealed that separation of homogenized emulsions at 250 bar and 1000 bar was due to
coalescence and aggregation, respectively. The initial PSD of the emulsions homogenized at
1000 bar was small, owing to this and the fact that the 1% pectin solutions used to prepare
these emulsions were more viscous, probably movement was limited thus less collision of
droplets. For CP-emulsions, when the DM increased from 16 to 95, decreasing stability was
observed (Figure 5.16A). This observation was complimented by the PSD plots which
depicted larger average particle sizes of emulsions at 250 bar compared to those at 1000 bar
with greater %volume of the emulsions at 1000 bar (Figure 5.16B). As smaller droplets are
more likely to be stable compared to larger ones, emulsions homogenized at 1000 bar were
more stable.
B) Pectin-protein emulsions
A similar influence of pressure was observed for the pectin-protein emulsions of both pectin
types, with decreasing average particle size as the applied homogenization pressure
increased as shown in Figure 5.17. In addition, owing to pressure-induced unfolding of
protein molecules and further biopolymer-biopolymer interactions in the presence of
polysaccharides such as pectin, could have strongly influenced adsorption of the pectin onto
the oil droplets surface hence the formation of smaller droplet sizes as pressure increased
(Dickinson and James, 2000).
After storage, both pectic-protein emulsions of both pectin sources depicted slight increase
in droplet size as shown in Figure 5.17.
74
0
2
4
6
8
10
12
0.1 1 10 100 1000
%V
olu
me
Particle diameter (µm)
0.1% - pH 3.0
250 bar
0
2
4
6
8
10
12
0.1 1 10 100 1000%
Vo
lum
eParticle diameter (µm)
0.1% - pH 3.0
1000 bar
Before storage
Figure 5.17: Particle size distribution plots of the pectin-protein emulsions of citrus and sugar beet
pectin at different homogenization pressures for a given pH and pectin concentration before storage.
5.4.4 Effect of storage temperature on stability of ultra-high pressure homogenized
emulsions
A) Pectin-emulsions
Looking at the stability images of any given DM, pectin concentration or pH (citrus pectin
emulsions), faster phase separation was observed at the highest storage temperature (35 °C)
compared to the lower temperatures (20 °C and 4 °C) after two weeks of storage (Figure
5.18A). The emulsions at the lowest temperature exhibited a lower degree of phase
separation. Higher temperatures increase the thermal energy of the whole emulsion system
leading to loss of entanglement of the conformational structure of the viscous emulsion (Kar
and Arslan, 1999; Milani and Maleki, 2012). Owing to this, oil droplets will easily diffuse
through the solution and come more in contact with each other leading to creaming or
coalescence or aggregation, all which are mechanisms that may cause emulsion
destabilization (Rao, 2013). Furthermore, at 35 °C for the citrus pectin emulsions, slightly less
phase separation was observed at pH 4.2 compared to that at pH 6.2. On the other hand, the
sugar beet pectin emulsions appeared more stable at the different pHs (Figure 5.18A). In
relation to the PSD plots, the PSD patterns only shifted slightly to the right indicating only
75
4 °C (0.5%)
2 W
pH 4.2
pH 6.2
35 °C (0.5%)
2W
pH 4.2
pH 6.2
CP 16 CP 65 CP 95 SBP 35 SBP 60CP 35
Figure 5.18: (A) Emulsion stability study images of homogenized pectin-emulsions of citrus and sugar
beet pectin at different pHs after two weeks of storage at 4 °C and 35 °C. Homogenization pressure =
500 bar.
76
After storage
Figure 5.18: (B) Particle size distribution curves of the pectin-emulsions of citrus and sugar beet
pectin at different pHs after two weeks of storage at 35 °C. Homogenization pressure = 500 bar.
slight increase in particle size while the citrus pectin emulsions depicted an increase in
%volume fraction and average particle size after storage (Figure 5.18B).
B) Pectin-protein emulsions
Similarly, the pectin-protein emulsions depicted phase separation in both pectin types after
storage under the highest storage temperature (35 °C) with more pronounced separation in
the citrus pectin samples. PSD plots also affirmed this with sugar beet and citrus pectin-
protein emulsions of similar DMs showing a shift to the right (towards the larger particle
sizes).
Generally, from the citrus and sugar beet pectin emulsions stability study, increasing pectin
concentration increased the stability of emulsions at a given DM and pH. Emulsion stability
also increased with increasing homogenization pressure accompanied by decreasing DM. On
the other hand, high temperature as well as with prolonged storage periods promoted
emulsion destabilization. Regarding the pectin emulsions, CP 16 showed the best stability for
the CP samples whereas both SBP samples depicted approximately similar stabilities.
Addition of protein in the preparation of protein-pectin emulsions did not exert any
improvement in stability of both CP and SBP samples.
77
In conclusion, the sugar beet pectin samples exhibited less physical separation, hence
greater stability than the citrus pectin samples. This may be due to structural differences in
these pectin sources. The better emulsifying and stabilizing effects of SBP may be attributed
its high acetyl and protein content in addition to high degree of branching (neutral sugars)
and polymerization (Jung and Wicker, 2012). SBP was still a better emulsifier than CP with
lower neutral sugar content. The poor emulsifying and stabilizing properties of CP are
probably due to low molecular weight, low protein and low acetylation content, low neutral
sugars content as well as low ferulic acid content.
78
6 GENERAL CONCLUSION
Extracted pectin is important in the food industry owing to its ability to improve structural
and functional properties of plant-based products. Its health benefits make it an even more
preferred functional food ingredient in several applications aimed at producing safe, healthy
and convenient foods. Although extracted pectin is mainly used in gelling, thickening and
stabilizing applications, the potential use of this polysaccharide as an emulsifying agent has
gained attention from some researchers and is the basis of this research.
The objective of this research was to investigate the effect of pectin structure on the
emulsifying capacity as well as the emulsion stabilizing capacity of citrus and sugar beet
pectins.
Commercially available citrus and sugar beet pectins with degrees of methylesterification
(DM) of 95% and 60%, respectively were structurally modified by incubating their watery
solutions for different periods in presence of carrot pectin methylesterase (PME). For citrus
pectin, the DMs of the resulting pectin were DM 65%, DM 35% and DM 16%, denoted as CP
16, CP 35 and CP 65, respectively. In the case of sugar beet pectin, a DM of 35% (denoted as
SBP 35) was obtained. The starting pectins (DM 95% and DM 60%, for CP and SBP,
respectively) were included as controls and denoted as CP 95 and SBP 60. Subsequent
characterization of various pectin structural properties (protein content, degree of
acetylation, degree of feruloylation, galacturonic acid, neutral sugars and molar mass)
revealed that no differences in these properties were exhibited by pectin samples from the
same source. Investigation of the emulsifying and emulsion stabilizing potential of the pectin
samples showed variations.
When considering the possible stabilizing capacity of both pectin types, the viscosity of the
pectin solutions plays an important role. Therefore, the apparent viscosity of the pectin
solutions was measured and results showed that it decreased as the DM of pectin increased
at any given pH and pectin concentration. CP 95 remained more or less constant with
increasing pH. By increasing the pectin concentration a significant increase in apparent
viscosity of the solutions was observed. High pectin concentrations being more concentrated
79
led to restricted movement within the solution and this enhanced the viscosity of the
solution. LM pectin solutions (CP 16 and CP 35) exhibited a shear thinning behavior which
was more pronounced as pH increased. Possibly, the loss in the network entanglement in LM
citrus pectin led to the significant influence of increasing shear rate on the viscosity of the
pectin solutions. The HM citrus pectin as well as the sugar beet pectin solutions exhibited
more or less a Newtonian behavior. Comparing the viscosities of the citrus and sugar beet
pectin solutions of a similar DM, i.e. CP 35 to SBP 35, SBP 35 showed a higher viscosity than
CP 35. Probably, acetyl groups which can lower the hydrophobicity of pectin leading to an
increase in the solubility of pectin facilitated the modification of the viscosity of the aqueous
medium.
Considering that the emulsifying potential can is attributed to the ability of an emulsifier (in
this case pectin) to adsorb onto the oil droplet surface in an emulsion, adsorbing capacity
was examined. For the citrus pectin samples, pectin with the lowest DM (CP 16) depicted the
largest droplet sizes as compared to CP 95 which had the smallest droplet sizes. A similar
trend was observed in SBP emulsions. Increasing pectin concentration increased the droplet
sizes in both CP and SPB emulsions. In citrus pectin, high DM pectin exhibited better
emulsifying potential than low DM pectin. Finally, the sugar beet pectin emulsions generally
depicted smaller droplet sizes compared to citrus pectin emulsions of a similar DM. Based on
the smaller droplet sizes of sugar beet pectin emulsions, this pectin type showed better
emulsifying properties than citrus pectin emulsions since the strong adsorption of a better
emulsifier gives rise to smaller droplets.
Stability studies on the emulsions prepared with watery pectin solutions showed that
stability increasing with decreasing storage temperature (35 °C, 20 °C and 4 °C). Results at 35
°C were presented in detail and these represent stability studies under accelerated
conditions. Before storage of citrus and sugar pectin emulsions, the initial droplet
size/particle size increased with increasing DM, pH as well as pectin concentration. In
addition, increasing homogenization pressure decreased the particle sizes of the pectin
emulsions at a given pectin concentration and pH. Storage temperature significantly
80
influenced the emulsion stability with a high degree of phase separation being observed at
35 °C compared to the lower temperatures 20 °C and 4 °C with sugar beet pectin emulsions
appearing more stable than the citrus pectin emulsions. The increase in thermal energy of
the whole system (emulsion) due to the high temperature may have caused greater degree
of phase separation. Similar trends were observed in the pectin-protein emulsions of both
pectin types prior to storage.
In general, although high DM pectin was better adsorbed onto the oil droplet surfaces at the
start of storage, it did not exhibit good emulsion stabilizing properties during storage.
Instead, it was the low DM pectin that exhibited better stabilizing properties. On the other
hand, sugar beet pectin samples portrayed better emulsifying and stabilizing capacity than
the citrus pectin samples as seen from images, microscopy and particle size distribution.
Although the presence of higher neutral sugars in sugar beet is reported to reduce its
thickening, gelling and stabilizing effects. Perhaps, the effect of high molecular weight, high
acetyl groups as well as high protein content was more effective than the downside of high
neutral sugars, leading to better emulsifying and stabilizing potential of sugar beet pectin.
Stabilization of the sugar beet pectin emulsions was further improved by increasing the
concentration of the emulsifier (pectin), reducing pH and increasing the homogenization
pressure.
It can thus be concluded that pectin structure influences the emulsifying and stabilizing
capacity of pectin.
This research presented relevant information on the effect of some processing parameters
on the functionality of citrus and sugar beet pectins. It also established some functional
properties with specific processing parameters which can form a basis for optimization of
some emulsion-based foods.
i
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