In quite remarkable as other choice of precipitants

In this
study, a preparation for the immobilization of fructosyltransferase using a
carrier-free technique was reported. The statistical approach to optimization
is of immense industrial importance especially for scale up process. Upon
extensive literature review, there is little or no information regarding the
use of a carrier free immobilization technique for fructosyltransferase. Hence,
our study is the first of its kind in that regard.

During the screening for the best precipitant
to use, we focused on common protein precipitants namely ammonium sulphate,
acetone and ethanol at concentration range of 40 to 70% (v/v). In the course of
the aggregation, ammonium sulphate was found to be the best precipitant. This
was not surprising as ammonium sulphate is regarded as the best in terms of
salting out of protein. Toluene wasn’t far behind, as it compared favourably
with ammonium sulphate. In the course of this study, both toluene and ethanol
were not able totally precipitate the enzyme prior to cross-linking with
glutaraldehyde. Hence, they had lower recovery when compared with ammonium
sulphate. Also, under repeated catalysis, the CLEA produced using ammonium
sulphate had a higher recovery after four rounds than toluene and ethanol. This
is suggestive of the complete aggregation observed when ammonium sulphate is
the cross-linker compared to the organic solvents. There was a gradual loss in
activity after as the rounds of catalysis increased. This might be as a result
of perhaps bioaccumulation of the products of sucrose catalysis. The ammonium
sulphate-CLEA was able to maintain its 100% residual activity over the four
rounds of catalysis. This is quite remarkable as other choice of precipitants
such as toluene and ethanol lost more than 50% and 90% respectively. Also, in
the initial choice of cross-linker concentration, it was observed that a high glutaraldehyde
concentration, up to 10% had very negligible adverse effect of CLEA catalysis.
The amount of glutaraldehyde reported in recent literature was 0.125% v/v
(Ortiz-Soto et al. 2009), we noted
that at this concentration, there wasn’t CLEA formation and bio catalysis was
very poor (result not shown). The high amount of glutaraldehyde had been
reported to inhibit the catalysis by CLEA because of the adverse effect of
excessive cross-linking. But we postulate that since glutaraldehyde reacts
primarily with the ?-amino groups of lysine and other groups such as hydroxyl,
secondary amino and alpha-amino (Migneault et al. 2004), the A. pullulans
fructosyltransferase might contain fewer lysine residues hence the need for a
high amount of glutaraldehyde.

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

In this study, statistical optimal
operational conditions were designed for CLEA bio catalysis as well as product
release. The effects of pH, concentration of glutaraldehyde and effective temperature
were varied using response surface methodology. In this Box Behnken model, it
was observed that reactions that involved generally low glutaraldehyde
concentrations had low aggregation yield irrespective of the pH and the
temperature (Table 3). Hence, this confirms our theory that perhaps fewer
lysine residues are present for cross-linking of the aggregates. The analysis
of variance (ANOVA) data revealed the model was statistically significant and
the lack of fit was not significant (Table 4). The Coefficient of variation, R2,
adeq Precision, Pred-R2 are 35.34, 0.94, 9.34 and -0.0035
respectively. The regression equation showing this interaction is given in
coded units as

Aggregation Yield  =
+197.80 +27.25 * A +9.87 * B +35.88* C -38.27 * A2 -91.53 * B2 -97.03 * C2                               +5.50* A * B +15.00* A * C +3.75 * B * C,

Where A, B and C represents pH, temperature
and glutaraldehyde concentration respectively.

The three-dimensional surface plots showed
varying degrees of interaction between the variables considered. The responses
obtained from the 3-D response surface plots indicate how the numerous
interactions that occur between the studied variables affect the aggregation
yield and it is very needful in characterizing the responses (Ademakinwa et al. 2017). The optimum conditions
statistically derived from the RSM model were 5% glutaraldehyde, pH 5.5 and 320C

In the response plots of the effects of pH
and temperature, it was observed from the shape of the curves that an increase
in pH led to an increase in the aggregation yield up to a point before a
decrease was observed and the same observation was observed as the temperature
was simultaneously increased. At higher temperature, the concomitant effects on
the aggregation yield was adverse.

The effects of glutaraldehyde concentration
and pH on the aggregation yield resulted in a bell-shaped curve indicating the
effects of increasing any of the variables on the aggregation yield. Initial
increase of glutaraldehyde increased the activation yield, further increase
over 5% resulted in loss of aggregation yield. The same was observed for the
effects of pH, in which increasing the pH resulted in the initial increase in
the aggregation yield but further increase did not lead to a significant loss
of the aggregation yield. The effect of pH showed perhaps a more moderate
effect as the pH increased.

The effect of glutaraldehyde and temperature
on the aggregation yield showed a much different interaction compared with the
combined variables discussed above. Initial increase to the central values led
to increase in aggregation yield but further increase above these central
values led to a significant decrease in aggregation yield. The use of the CLEA
under the obtained optimized conditions resulted in the use of the CLEA in
repeated catalysis over 6 catalytic cycles still retaining about 70% of its
residual activity. This is albeit less compared with the ten catalytic cycles
obtained when Bacillus subtilis
levansucrase was used (Ortiz-Soto et al.
2009).

The SEM/EDX of the CLEA showed the morphology
as well as elemental composition of the CLEA. The size of the nanoparticle
ranges from 10 to 40 µm and it wasn’t evenly shaped. The elemental composition
revealed that the CLEA had a higher composition of phosphorus, sulphur and
oxygen. The shape of the CLEA increased after quenching and repeated catalysis.
The microscopic analysis of the CLEA revealed that the size to be about 10 µm
while post quenching, the size increased and it was well close to 40 µm. Ortiz-Soto
et al. (2009) reported the effect of
quenching on the sizes of the CLEA. Increased sizes were also noted by the
levansucrase CLEA after quenching and repeated catalysis.

To further ascertain structural features between
free enzyme and CLEAs, the secondary structure of all CLEAs was observed by
FT-IR spectroscopy this is because FTIR is considered an important technique to
monitor conformational changes in proteins (Jung, 2000). The structural
variation in the protein’s conformation is mostly studied in the range that constitutes
the amide I band as this is the most intense absorption band for polypeptides
(Susi and Byler, 1986).  It was observed that, there as increased
?-sheet component in the cross-linked enzyme aggregate compared to the free
enzyme. It is thus concluded that cross-linking with glutaraldehyde allowed for
the transformation of helical structures and beta sheet to beta turns due to
protein aggregation

The transfructosylation of sucrose did yield
fructooligosaccharides by the CLEA. The FTIR spectra of the aqueous solution of
each pure sugar component as well as the purified reaction products is shown in
as shown in figure 8.  The characteristic
carbohydrate fingerprint regions from the FTIR spectra for glucose, sucrose,
commercial fructooligosaccharides, fructose and purified reaction products FOS
had peaks at regions that varied from 1007 to 1193 cm-1, 995-1047 cm-1,
969 to 1039 cm-1, 956-1171 cm-1 and 968-1271 cm-1.
The crude reaction products had peaks at 969, 997.2, 1078 and 1095 cm-1.
It was observed that the spectral signatures of each sugars were
distinguishable. Sugars are known to have exocyclic and endocyclic C-O bonds
located at 995 cm-1 and 1080 cm-1 for sucrose and
glucose/fructose respectively. The broad band observed around 3700 to 3000 cm-1
are fingerprint regions for water and -OH absorption frequencies. There is the
presence of other fingerprint regions for other functional groups such as peaks
that corresponds to stretching vibrations of – C-H inside CH3 or CH2
groups. Characteristically, a unique peak that exists for the
fructooligosaccharides was found in the 965 cm-1 regions. This peak
was found in the FTIR spectra of the commercial FOS, crude and purified
reaction products and it was noticeably absent in sucrose and glucose. This is
a non-destructive and non-toxic confirmation of the transfructosylation of
sucrose to fructooligosaccharides by the CLEA.

The physiological and biotechnological
importance of FOS necessitated the investigations of the use of FOS from A. pullulans NAC8 as a prebiotic source.
There is the increasing trend for the addition of fructooligosaccharides into
food products because they have prebiotic properties (Bali et al., 2015, Flores-Maltos et
al., 2015). Their consumption increases fecal bolus and the frequency of
depositions and also reduce constipation which is regarded as a problem in the
modern world and also in infants (early months) (Sabater-Molina et al., 2009).

In this study, strains
that fermented FOS (and possibly produced acid end products) grew as colonies
surrounded by a yellow zone against a purple background. The use of
bromocresol purple as an indicator of utilization of FOS by probiotic strains
served as a necessary yardstick needed for quick preliminary screening of these
strains. As reported in this study, the newly synthesized FOS as well as the
commercially obtained FOS both had colonies that existed as a yellow zone
against a yellow background (Plate 4.3). This observation was similar to the
report of Kaplan and Hutckins, (2000), where several Lactobacillus sp. were able to utilize FOS concomitantly forming
yellow colonies.

The prebiotic effectiveness of A. pullulans
FOS on a probiotic strain, Lactobacillus sp. revealed that 1% (w/v) FOS had a
slight stimulatory effect on bi?dobacterial growth compared with the controlled
medium also the pH values of the media dropped with the increase of the mass of
bi?dobacteria (Figure 4.79). The growth curves of Lactobacillus sp. reached plateau phase after 24 h cultivation with
FOS (Figure 4.80), whereas it took 36 h to reach plateau phase without FOS
(Figure 4.81). Therefore, the growth rate of the bacteria in FOS-containing
medium was faster than that in the medium without FOS.
Moreover, the ?nal bacterial mass in the medium with FOS was greater than that
in the medium without FOS. pH values of the media dropped along with the
increases of bene?cial bacterial populations.

During the fermentation of fructose
oligomers by probiotic bateria, a gradual decrease in the pH is often observed and
it is indicative of the production of lactic acid, hence the decrease in pH (Rastall
et al., 2000). The decrease of pH
values after incubation with FOS suggests that the bi?dobacteria were able to
utilize FOS. These results indicated that FOS potently stimulated the growth of
bi?dobacteria, which supported the potential prebiotic effect of FOS.

The
FOS produced by A. pullulans in this
study, was able to enhance the growth of prebiotic bacteria that is native to
the gastrointestinal microbiota, hence, the use of these FOS as food additives
is advised. This is because eaten foodstuffs are usually fortified with
prebiotic ingredients, such as inulin and oligo-fructose (Holscher, 2017).

In this
study, a preparation for the immobilization of fructosyltransferase using a
carrier-free technique was reported. The statistical approach to optimization
is of immense industrial importance especially for scale up process. Upon
extensive literature review, there is little or no information regarding the
use of a carrier free immobilization technique for fructosyltransferase. Hence,
our study is the first of its kind in that regard.

During the screening for the best precipitant
to use, we focused on common protein precipitants namely ammonium sulphate,
acetone and ethanol at concentration range of 40 to 70% (v/v). In the course of
the aggregation, ammonium sulphate was found to be the best precipitant. This
was not surprising as ammonium sulphate is regarded as the best in terms of
salting out of protein. Toluene wasn’t far behind, as it compared favourably
with ammonium sulphate. In the course of this study, both toluene and ethanol
were not able totally precipitate the enzyme prior to cross-linking with
glutaraldehyde. Hence, they had lower recovery when compared with ammonium
sulphate. Also, under repeated catalysis, the CLEA produced using ammonium
sulphate had a higher recovery after four rounds than toluene and ethanol. This
is suggestive of the complete aggregation observed when ammonium sulphate is
the cross-linker compared to the organic solvents. There was a gradual loss in
activity after as the rounds of catalysis increased. This might be as a result
of perhaps bioaccumulation of the products of sucrose catalysis. The ammonium
sulphate-CLEA was able to maintain its 100% residual activity over the four
rounds of catalysis. This is quite remarkable as other choice of precipitants
such as toluene and ethanol lost more than 50% and 90% respectively. Also, in
the initial choice of cross-linker concentration, it was observed that a high glutaraldehyde
concentration, up to 10% had very negligible adverse effect of CLEA catalysis.
The amount of glutaraldehyde reported in recent literature was 0.125% v/v
(Ortiz-Soto et al. 2009), we noted
that at this concentration, there wasn’t CLEA formation and bio catalysis was
very poor (result not shown). The high amount of glutaraldehyde had been
reported to inhibit the catalysis by CLEA because of the adverse effect of
excessive cross-linking. But we postulate that since glutaraldehyde reacts
primarily with the ?-amino groups of lysine and other groups such as hydroxyl,
secondary amino and alpha-amino (Migneault et al. 2004), the A. pullulans
fructosyltransferase might contain fewer lysine residues hence the need for a
high amount of glutaraldehyde.

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

In this study, statistical optimal
operational conditions were designed for CLEA bio catalysis as well as product
release. The effects of pH, concentration of glutaraldehyde and effective temperature
were varied using response surface methodology. In this Box Behnken model, it
was observed that reactions that involved generally low glutaraldehyde
concentrations had low aggregation yield irrespective of the pH and the
temperature (Table 3). Hence, this confirms our theory that perhaps fewer
lysine residues are present for cross-linking of the aggregates. The analysis
of variance (ANOVA) data revealed the model was statistically significant and
the lack of fit was not significant (Table 4). The Coefficient of variation, R2,
adeq Precision, Pred-R2 are 35.34, 0.94, 9.34 and -0.0035
respectively. The regression equation showing this interaction is given in
coded units as

Aggregation Yield  =
+197.80 +27.25 * A +9.87 * B +35.88* C -38.27 * A2 -91.53 * B2 -97.03 * C2                               +5.50* A * B +15.00* A * C +3.75 * B * C,

Where A, B and C represents pH, temperature
and glutaraldehyde concentration respectively.

The three-dimensional surface plots showed
varying degrees of interaction between the variables considered. The responses
obtained from the 3-D response surface plots indicate how the numerous
interactions that occur between the studied variables affect the aggregation
yield and it is very needful in characterizing the responses (Ademakinwa et al. 2017). The optimum conditions
statistically derived from the RSM model were 5% glutaraldehyde, pH 5.5 and 320C

In the response plots of the effects of pH
and temperature, it was observed from the shape of the curves that an increase
in pH led to an increase in the aggregation yield up to a point before a
decrease was observed and the same observation was observed as the temperature
was simultaneously increased. At higher temperature, the concomitant effects on
the aggregation yield was adverse.

The effects of glutaraldehyde concentration
and pH on the aggregation yield resulted in a bell-shaped curve indicating the
effects of increasing any of the variables on the aggregation yield. Initial
increase of glutaraldehyde increased the activation yield, further increase
over 5% resulted in loss of aggregation yield. The same was observed for the
effects of pH, in which increasing the pH resulted in the initial increase in
the aggregation yield but further increase did not lead to a significant loss
of the aggregation yield. The effect of pH showed perhaps a more moderate
effect as the pH increased.

The effect of glutaraldehyde and temperature
on the aggregation yield showed a much different interaction compared with the
combined variables discussed above. Initial increase to the central values led
to increase in aggregation yield but further increase above these central
values led to a significant decrease in aggregation yield. The use of the CLEA
under the obtained optimized conditions resulted in the use of the CLEA in
repeated catalysis over 6 catalytic cycles still retaining about 70% of its
residual activity. This is albeit less compared with the ten catalytic cycles
obtained when Bacillus subtilis
levansucrase was used (Ortiz-Soto et al.
2009).

The SEM/EDX of the CLEA showed the morphology
as well as elemental composition of the CLEA. The size of the nanoparticle
ranges from 10 to 40 µm and it wasn’t evenly shaped. The elemental composition
revealed that the CLEA had a higher composition of phosphorus, sulphur and
oxygen. The shape of the CLEA increased after quenching and repeated catalysis.
The microscopic analysis of the CLEA revealed that the size to be about 10 µm
while post quenching, the size increased and it was well close to 40 µm. Ortiz-Soto
et al. (2009) reported the effect of
quenching on the sizes of the CLEA. Increased sizes were also noted by the
levansucrase CLEA after quenching and repeated catalysis.

To further ascertain structural features between
free enzyme and CLEAs, the secondary structure of all CLEAs was observed by
FT-IR spectroscopy this is because FTIR is considered an important technique to
monitor conformational changes in proteins (Jung, 2000). The structural
variation in the protein’s conformation is mostly studied in the range that constitutes
the amide I band as this is the most intense absorption band for polypeptides
(Susi and Byler, 1986).  It was observed that, there as increased
?-sheet component in the cross-linked enzyme aggregate compared to the free
enzyme. It is thus concluded that cross-linking with glutaraldehyde allowed for
the transformation of helical structures and beta sheet to beta turns due to
protein aggregation

The transfructosylation of sucrose did yield
fructooligosaccharides by the CLEA. The FTIR spectra of the aqueous solution of
each pure sugar component as well as the purified reaction products is shown in
as shown in figure 8.  The characteristic
carbohydrate fingerprint regions from the FTIR spectra for glucose, sucrose,
commercial fructooligosaccharides, fructose and purified reaction products FOS
had peaks at regions that varied from 1007 to 1193 cm-1, 995-1047 cm-1,
969 to 1039 cm-1, 956-1171 cm-1 and 968-1271 cm-1.
The crude reaction products had peaks at 969, 997.2, 1078 and 1095 cm-1.
It was observed that the spectral signatures of each sugars were
distinguishable. Sugars are known to have exocyclic and endocyclic C-O bonds
located at 995 cm-1 and 1080 cm-1 for sucrose and
glucose/fructose respectively. The broad band observed around 3700 to 3000 cm-1
are fingerprint regions for water and -OH absorption frequencies. There is the
presence of other fingerprint regions for other functional groups such as peaks
that corresponds to stretching vibrations of – C-H inside CH3 or CH2
groups. Characteristically, a unique peak that exists for the
fructooligosaccharides was found in the 965 cm-1 regions. This peak
was found in the FTIR spectra of the commercial FOS, crude and purified
reaction products and it was noticeably absent in sucrose and glucose. This is
a non-destructive and non-toxic confirmation of the transfructosylation of
sucrose to fructooligosaccharides by the CLEA.

The physiological and biotechnological
importance of FOS necessitated the investigations of the use of FOS from A. pullulans NAC8 as a prebiotic source.
There is the increasing trend for the addition of fructooligosaccharides into
food products because they have prebiotic properties (Bali et al., 2015, Flores-Maltos et
al., 2015). Their consumption increases fecal bolus and the frequency of
depositions and also reduce constipation which is regarded as a problem in the
modern world and also in infants (early months) (Sabater-Molina et al., 2009).

In this study, strains
that fermented FOS (and possibly produced acid end products) grew as colonies
surrounded by a yellow zone against a purple background. The use of
bromocresol purple as an indicator of utilization of FOS by probiotic strains
served as a necessary yardstick needed for quick preliminary screening of these
strains. As reported in this study, the newly synthesized FOS as well as the
commercially obtained FOS both had colonies that existed as a yellow zone
against a yellow background (Plate 4.3). This observation was similar to the
report of Kaplan and Hutckins, (2000), where several Lactobacillus sp. were able to utilize FOS concomitantly forming
yellow colonies.

The prebiotic effectiveness of A. pullulans
FOS on a probiotic strain, Lactobacillus sp. revealed that 1% (w/v) FOS had a
slight stimulatory effect on bi?dobacterial growth compared with the controlled
medium also the pH values of the media dropped with the increase of the mass of
bi?dobacteria (Figure 4.79). The growth curves of Lactobacillus sp. reached plateau phase after 24 h cultivation with
FOS (Figure 4.80), whereas it took 36 h to reach plateau phase without FOS
(Figure 4.81). Therefore, the growth rate of the bacteria in FOS-containing
medium was faster than that in the medium without FOS.
Moreover, the ?nal bacterial mass in the medium with FOS was greater than that
in the medium without FOS. pH values of the media dropped along with the
increases of bene?cial bacterial populations.

During the fermentation of fructose
oligomers by probiotic bateria, a gradual decrease in the pH is often observed and
it is indicative of the production of lactic acid, hence the decrease in pH (Rastall
et al., 2000). The decrease of pH
values after incubation with FOS suggests that the bi?dobacteria were able to
utilize FOS. These results indicated that FOS potently stimulated the growth of
bi?dobacteria, which supported the potential prebiotic effect of FOS.

The
FOS produced by A. pullulans in this
study, was able to enhance the growth of prebiotic bacteria that is native to
the gastrointestinal microbiota, hence, the use of these FOS as food additives
is advised. This is because eaten foodstuffs are usually fortified with
prebiotic ingredients, such as inulin and oligo-fructose (Holscher, 2017).

x

Hi!
I'm Mary!

Would you like to get a custom essay? How about receiving a customized one?

Check it out