3.1Physical and chemical characterization:
3.1.1 Moisture content and water activity
The final powder reached low moisture values (12.75 % w.b.), which led to water activity levels that ensure microbiological stability (aw=0.58). Regarding the sorption characteristics of the cladode powder, there are large deviations in the available literature. Sáenz et al. (2010) reported water activity 0.53 for cladode powder with moisture 7.14 % w.b., while Boukid et al. (2015) reported water activities 0.19 – 0.36 for three powders dried at different temperatures, having a moisture content 12.66 - 18.25 % w.b. The aforementioned results differ substantially with our findings. Harrak (2021) studied the sorption curves for powders derived from cladodes with different age (starting from less than 1 year up to 3.5 years) and found important variations among the different ages. Therefore, it seems that the sorption characteristics vary widely. It should be mentioned that in our study the peel had been removed, hence altering the composition of the final powder. Peel has a different composition than cladode pulp (Sepúlveda et al., 2013), which could affect the sorption curves.
3.1.2 Dietary fiber and mucilage molecular weight
Cladode powder properties are presented in Table 2. High amounts of dietary fiber were determined, exceeding 50% in total solids, while the IDF:SDF ratio was strongly in favor of the former. It has been documented that this ratio increases with the aging of the cladode(Rodríguez-Garcia et al., 2007, Contreras-Padilla et al., 2012, Hernández-Urbiola et al., 2015). In the previous studies a clear trend has been observed, where the soluble fraction exceeds 25% d.b. at young age and decreases as low as 10% d.b. with aging. In addition, Sepúlveda et al.(2013) studied the differences between whole and peeled cladodes and found that peeling led to a decrease in the SDF fraction. These two factors (age > 4 months and peeling) probably explain the low concentration of SDF in our sample (8.94 % d.b.).
Extracted mucilage was found to have a molecular weight of 2.18 106Da., which is very similar to the values reported from Rodríguez-González et al. (2021), where the Mw ranged from 2.3 106 up to 11.9 106 Da, depending on the age of the cladode. The results of Trachtenberg & Mayer (1982) were in the same order of magnitude (4.3 106Da). However, much lower values (approx. 103-104 Da) have been reported by other researchers (Espino-Díaz et al., 2010, L. Medina-Torres et al., 2000).
Table 2: Physical and chemical properties of cladode powder.
Composition, density and color
|
Mean ± SD
|
Moisture (% w.b.)
|
12.75 ± 0.38
|
Total Dietary Fiber (% d.b.)
|
51.02 ± 0.94
|
Insoluble Dietary Fiber (% d.b.)
|
42.08 ± 0.68
|
Soluble Dietary Fiber (% d.b.)
|
8.94 ± 0.66
|
Insoluble : Soluble Dietary Fiber
|
4.71
|
Available Carbohydrates (% d.b.)
|
29.65 ± 2.33
|
Water activity
|
0.58 ± 0.01
|
Tap density (g/ ml)
|
0.62 ± 0.01
|
Color
|
|
L
|
76.02 ± 0.29
|
a*
|
3.05 ± 0.04
|
b*
|
21.04 ± 0.21
|
Values are shown as mean ± standard deviation, n =3.
3.1.3 Color stability
The color parameters of the cladode powder throughout the storage period are shown in Fig. 1. The initial powder bore an appealing green color (L = 76.02, a* = -3.05, b* = 21.04), despite the removal of the peels. The peel is the part that has the greenest color, since it is responsible for photosynthesis, therefore peeling probably affects the color of the final powder. To our knowledge, there are no studies reporting color of peeled cladode powder. It has also been documented that the color of cladode powder also depends on the age of the cladode, turning paler as the age increases (Harrak, 2021).
Powder color degraded when stored in an airtight container in the dark, which is expressed with a reduction in parameter b* (Fig.1c). However, an acceptable color has been retained (Figure 1d). On the other hand, powder exposed to light exhibited greater degradation, leading to a substantially altered appearance. The degradation is expressed not only by a more intense reduction in the parameter b*, but in an increase in parameter a* as well (-3.21 and -0.77 for powder stored in dark and light, respectively). Negative a* values express the green color, so this increase is indicative of the decolorization, which is observed. Consequently, protection from light is a crucial factor for maintaining cladode powder characteristic color.
3.2 Rheological characterization
3.2.1 Aqueous suspensions
All samples exhibited pseudoplastic behavior. Consistency coefficient of cladode suspensions were 0.051 and 0.059 Pa sn for pH 4 and 6, respectively (Table 3). Luis Medina-Torres et al. (2011) reported values 0.17 - 0.34 for similar concentration (3% w/v) of cladode powder dried at 45oC. Chaloulos et al. (2021) also found higher values for 5% w/w cladode suspensions (0.15 – 0.20 Pa sn) for partially vacuum dried cladodes. The highest values for cladode powder suspensions have been reported by Ramírez-Moreno et al. (2013) both for raw and boiled cladodes at a concentration 3.2% (4.07 – 6.37 and 2.66 – 3.53 Pa sn, respectively). The lower values in the present study may be attributed to several factors. Age (Contreras-Padilla et al., 2016) and cultivar (Du Toit et al., 2019, López-Palacios et al., 2016) are known to affect the rheological characteristics of cladode. A substantial difference in our study is that we used peeled cladodes. Majdoub et al., (2001)found that polysaccharides extracted from pulp exhibited increased viscosity compared to polysaccharides from cladode peels. However, in our study low viscosity is observed.
Table 3: Power law parameters for aqueous suspensions (C: 3% cladode, C-Cas: 3% cladode + 1 % sodium caseinate, C-WPI: 3% cladode + 1 % whey protein isolate, C-Gel: 3% cladode + 1% gelatin, CaCl2 and NaCl: 3% w/w cladode powder aqueous suspensions, with CaCl2 or NaCl addition for ionic strength adjustment at I = 1.45 M).
pH
|
Sample
|
K (Pa sn)
|
n
|
R2
|
RMSE*
|
pH = 6
|
C
|
0,059
|
0,61
|
0,991
|
0,045
|
C -Cas
|
0,151
|
0,49
|
0,992
|
0,135
|
C-WPI
|
0,065
|
0,61
|
0,971
|
0,100
|
C-Gel
|
0,482
|
0,26
|
0,965
|
0,215
|
pH = 4
|
C
|
0,051
|
0,59
|
0,941
|
0,035
|
C -Cas
|
0,081
|
0,51
|
0,994
|
0,063
|
C-WPI
|
0,187
|
0,44
|
0,999
|
0,571
|
C-Gel
|
0,028
|
0,60
|
0,963
|
0,023
|
|
CaCl2
|
0,112
|
0,51
|
0,988
|
0,411
|
|
NaCl
|
0,147
|
0,49
|
0,980
|
0,365
|
*Root mean square error
The effects of protein addition were pH dependent (Fig.2). In pH 6 GEL addition increased apparent viscosity values almost an order of magnitude. It should be noted that solutions of plain gelatin at the same concentration (1% w/w), prepared in the same way, had viscosity similar with pure water (data not shown). Therefore, the increase in viscosity can be attributed to interactions between gelatin and cladode powder. CAS had a less potent effect, while WPI didn’t alter the rheological properties substantially. However, in pH 4 these trends were reversed. C-CAS exhibited similar behavior with C, while C-GEL had even lower values. On the other hand, C-WPI exhibited the greatest viscosity in this pH.
Cladode mucilage, which is responsible for the viscous properties of cladode powder is a polysaccharide, which is negatively charged at pH 2-10, being more negatively charged at pH 6 rather than pH 4 (Quinzio et al., 2018). The three proteins studied here are below their isoelectric point at pH 4, being positively charged, contrary to pH 6, where they are negatively charged. Therefore, at pH 6 proteins and mucilage are both negatively charged, possibly favoring repulsive interactions between molecules. These repulsions are probably responsible for the more viscous character of C-CAS and C-GEL. WPI though did not appear to have any effect on viscosity at this pH, possibly because it is consisted mainly by globular proteins. This could mean that WPI has a weak effect in terms of both steric hindrance and repulsive interactions, as it is a more packed molecule. On the other hand, at pH 4 proteins and mucilage are oppositely charged, favoring electrostatic complexes to occur. Regarding gelatin, the formation of complex coacervates with cladode mucilage has been previously reported at pH below 5, where gelatin is positively charged (Otálora et al., 2019). The formation of such complexes probably indicate that mucilage is bound and cannot contribute as much to the thickening of the suspension.
3.2.2 Salt solutions
The effect of ions on the rheological properties of cladode powder aqueous suspensions was also evaluated. CaCl2 led to slightly lower viscosity values compared to NaCl (Figure 3), which is in agreement with Medina-Torres et al., (2000), who found that the presence of divalent cations leads to lower viscosity values for mucilage extracted from cactus cladodes. Du Toit et al. (2019) reported lower values for CaCl2 than NaCl for mucilage extracted from Algerian cactus cladodes but found no difference for the other cultivars tested in that study. On the other hand, Quinzio et al. (2018)reported a viscosity increase with CaCl2.Network formation by absorption of divalent Ca2+ was proposed. Calcium cations can act as a bridge between negatively charged groups, such as the ionized carboxyl groups, which promotes the gelling of low methoxy pectins (Fraeye et al., 2010, Thibault & Ralet, 2003). However, the effect of divalent cations on mucilage suspensions remains unclear. It must be noted that all the previous studies have used purified mucilage, while in our study whole cladode powder was used, which is already rich in calcium (McConn &Nakata, 2004). It can be concluded from our study that little differences were observed between monovalent and divalent cations.
3.2.3 Soup models
Soup models with cladode and/ or corn starch were prepared and their rheological properties were evaluated. Casson model exhibited worse fit than Power law model, apart from S3, in which the R2 values was similar for the two models (Table 4). S3 exhibited high viscosity values (K = 1.20 Pa sn), while its substitution with cladode powder gradually decreased the consistency coefficient down to 0.16 for C3. Rivera-Corona et al. (2014) studied the viscoelastic properties of sorghum starch gels, while substituting 5% and 10% of the starch with purified mucilage from cactus cladodes. They observed a substantial increase both in storage and loss modulus with 10% substitution, revealing a network enhancement with mucilage. In our study though, whole cladode powder was used, which would yield only 9% mucilage at most (our soluble fiber fraction was 8.94 % d.b.). This means that the addition of 1% cladode powder adds only 0.09% mucilage. Further increase in cladode powder concentration achieved higher values, resembling the S3 sample at a concentration of 8% w/w. Therefore, quite higher concentrations of cladode powder are needed for the same thickening effect. However, the thickening capacity of cladode powder is noticeable since the control soup model with no thickener had a viscosity similar to that of plain water (approximately 1.1 mPa s). On the other hand, when purified mucilage was used, consistency coefficient was diminished to 3*10-3 Pa*s (Table 4). Based on these results, cladode powder seems to be a more potent thickening agent compared to the corresponding concentration of purified mucilage. It’s hard to address the reason for such differences, but it is possible that the suspended particles in cladode powder suspensions interfere with the measurement. Another possible explanation is that the purification process (ethanol, drying) alters the rheological properties of the polysaccharide. It is also possible that other soluble compounds that may enhance the thickening capacity, such as proteins, remain soluble after the ethanol addition, hence are not isolated along with the mucilage.
Table 4: Power law and Casson model parameters for soup models (S3: 3% corn starch, S2C1: 2% corn starch + 1% cladode powder, S1C2: 1% corn starch + 2% cladode, C3: 3% cladode powder, C5: 5% cladode powder, C8: 8% cladode powder).
Power Law
|
pH
|
Sample
|
K (Pa sn)
|
n
|
R2
|
RMSE
|
pH = 5
|
C10
|
1,48
|
0,41
|
0,911
|
0,911
|
C8
|
1,13
|
0,40
|
0,995
|
0,076
|
C5
|
0,28
|
0,38
|
0,982
|
0,069
|
C3
|
0,16
|
0,52
|
0,972
|
0,021
|
C2
|
0,09
|
0,26
|
0,986
|
0,018
|
C1
|
0,01
|
0,58
|
0,852
|
0,008
|
S1C2
|
0,22
|
0,51
|
0,968
|
0,030
|
S1.5C1.5
|
0,26
|
0,44
|
0,991
|
0,057
|
S2C1
|
0,46
|
0,43
|
0,989
|
0,045
|
S3
|
1,20
|
0,47
|
0,992
|
0,184
|
S2
|
0,24
|
0,40
|
0,990
|
0,048
|
S1
|
0,03
|
0,47
|
0,969
|
0,014
|
S1M2
|
0,03
|
0,63
|
0,981
|
0,002
|
S1.5M1.5
|
0,13
|
0,46
|
0,969
|
0,009
|
S2M1
|
0,28
|
0,43
|
0,962
|
0,018
|
M3
|
0,003
|
1,00
|
0,979
|
0,000
|
pH = 4,3
|
C3
|
0,09
|
0,58
|
0,98139
|
0,01
|
pH = 5,0
|
C3
|
0,11
|
0,56
|
0,96724
|
0,01
|
pH = 6,2
|
C3
|
0,14
|
0,56
|
0,9727
|
0,02
|
Casson
|
pH
|
Sample
|
Kc(Pa0.5)
|
K0c (Pa0.5 s0.5)
|
R2
|
RMSE
|
pH = 5
|
C10
|
0,25
|
0,91
|
0,953
|
0,659
|
C8
|
0,20
|
0,82
|
0,983
|
0,61016
|
C5
|
0,10
|
0,40
|
0,997
|
0,0778
|
C3
|
0,13
|
0,24
|
0,879
|
0,07424
|
C2
|
0,03
|
0,26
|
0,948
|
0,0669
|
C1
|
0,03
|
0,05
|
0,891
|
0,0051
|
S1C2
|
0,15
|
0,29
|
0,862
|
0,11367
|
S1.5C1.5
|
0,12
|
0,37
|
0,986
|
0,07977
|
S2C1
|
0,16
|
0,48
|
0,932
|
0,240
|
S3
|
0,27
|
0,76
|
0,982
|
0,425
|
S2
|
0,09
|
0,37
|
0,994
|
0,085
|
S1
|
0,04
|
0,12
|
0,989
|
0,004
|
S1M2
|
0,07
|
0,14
|
1,000
|
0,002
|
S1.5M1.5
|
0,07
|
0,34
|
1,000
|
0,003
|
S2M1
|
0,09
|
0,51
|
1,000
|
0,006
|
M3
|
0,049
|
0,00
|
0,979
|
0,000
|
pH = 4,3
|
C3
|
0,12
|
0,16
|
0,868
|
0,038
|
pH = 5,0
|
C3
|
0,11
|
0,19
|
0,844
|
0,053
|
pH = 6,2
|
C3
|
0,13
|
0,22
|
0,867
|
0,063
|
*Root mean square error
**In the last triplet for pH, acetic acid is used instead of vinegar. The ionic strength was adjusted with NaCl addition at pH 5 and pH 6.2, in order to achieve equal ionic strength with pH 4.3, where the maximum acetic acid was added.
Power law model had a satisfactory fit for the consistency coefficient as a function of concentration (Fig.4C). It has been shown that the index b, representing the slope in logarithmic plot, varies for a given polysaccharide, depending on the concentration range. At low enough concentrations the slope is smaller, while a region of higher slope is expected at concentrations above a critical concentration, where entanglements are thought to be achieved (Doublier, 1981, Lopez et al., 2017, Potier et al., 2020). In the range tested in the present study, above a critical concentration of slope change, satisfactory correlation is observed, with starch exhibiting a greater dependence on concentration (b index 3.27 compared to 2.17 for cladode powder) and contributing more to the consistency of the soup as indicated by the greater slope of the curve.
Based on the logarithmic mixing law (Eq. 5) slightly negative deviations were observed (Fig. 5), therefore the underlying interactions, if any, are counterproductive in terms of thickening capacity. The samples with the purified mucilage exhibited very low viscosity and no substantial interaction was observed. However, our substitution was designed to be compared to the respective cladode concentration. Hussain et al.(2022) used either native or acetylated cactus cladode extract as a cactus mucilage source and studied the effect of corn starch substitution (2% and 5% of starch) on starch gels. All samples but one (2% acetylated mucilage) exhibited decreased final viscosity, which theoretically agrees with our findings. However, it must be noted that in our study the whole powder was used, which is rich in insoluble material. Rivera-Corona et al.(2014) reported substantial increase in storage and loss modulus of sorghum starch gels when cactus mucilage substituted the starch at 5 and 10%.
Regarding the effect of pH on the viscosity of the C3 soup system, a decreasing trend was observed when pH was lowered from 6.2 to 4.3. However, the differences were small with little importance (Fig. 6).