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Cgrygorczyk 2013
Cgrygorczyk 2013
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
a r t i c l e i n f o a b s t r a c t
Article history: The objective of this work was to compare the gelation of soymilk particles induced by the acidification of
Received 16 November 2012 a commercial starter culture with that resulting by addition of glucono-d-lactone (GDL). Structure
Received in revised form 24 March 2013 formation was followed using rheology, and the microstructure was observed by confocal microscopy.
Accepted 25 March 2013
Acidification of lactic acid bacteria resulted in a higher gelation pH (pH 6.29 ± 0.05) compared to that
Available online 25 April 2013
of a gel induced by GDL (pH 5.9 ± 0.04). This difference was attributed to the longer time available
for rearrangements of the soymilk particles in soymilk with starter cultures compared to the fast
Keywords:
acidification by GDL. In spite of the earlier gelation pH, there were no observed differences in the final
Soy protein
Lactic acid bacteria
gel stiffness measured at pH 5.1, the value of tan d, the frequency dependence and the linear viscoelastic
Glucono-d-lactone range of the gels measured at the final pH. Microstructural observations also showed a similar protein
Soymilk network structure.
Acid induced gel Ó 2013 Elsevier Ltd. All rights reserved.
0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.03.096
A. Grygorczyk, M. Corredig / Food Chemistry 141 (2013) 1716–1721 1717
& Chattopadhyay, 2011; Yang, Fu, & Li, 2012), no data exist in the light scattering (Mastersizer S; Malvern Instruments Inc.,
literature regarding the formation of the structure of soymilk acid- Southborough, MA), using 1.46 as the refractive index of the soy-
gels produced using lactic acid bacteria. milk particles and 1.333 as the refractive index of the dispersant
Several studies exist on the acid-induced aggregation of soymilk (water) as previously reported (Malaki Nik et al., 2009). The ratio
particles acidified with GDL (Kohyama et al., 1995; Malaki Nik of 11S:7S protein in the soy flour and soymilk was determined
et al., 2011; Tay & Perera, 2006). The gel pH ranges between 5.6 using SDS–PAGE, using conditions published in the literature
and 5.8 depending on the variety of soybean used, and different (Keerati-u-rai & Corredig, 2009). Gel analysis was carried out using
concentrations of GDL do not result in differences in the gelation a Gel Doc™ EZ Imager (Bio-Rad, Mississauga, ON, Canada). Crude
pH (Malaki Nik et al., 2011). However, acidification using lactic oil content of the soybeans was determined using the soxhlet oil
acid bacteria is known to be far slower than GDL acidification extraction method with petroleum ether (AOAC method 945.16)
(Lucey, Tamehana, Singh, & Munro, 1998). with a Labline multi-unit extraction heater (Barnstead Labline;
In consideration of the complex composition of soymilk parti- Thermo Scientific, Asheville, NC). The fat content of soymilk was
cles, it may be hypothesised that allowing more time for protein determined using the Babcock method (AOAC method 989.04,
rearrangements to occur may influence the gelation behaviour of 2000) with a Babcock System for Fat Analysis (Cole-Parmer, Mon-
soymilk. Although studies exist comparing the acidification of treal, QC, Canada) as previously reported in the literature (Buono,
cow’s milk using GDL or lactic acid cultures (Lucey et al., 1998), Erickson, Fung, & Jeon, 1990). Mineral analysis was determined
no such studies are reported for soymilk. Thus the purpose of this using inductively coupled plasma optical emission spectroscopy
study was to evaluate the difference in the gelation behaviour of by the advanced analytical laboratories at the University of Guelph
soymilk particles during acidification induced with GDL or bacte- (Guelph, Ontario, Canada).
rial culture.
2.3. Gelation experiments
To determine the amount of protein as well as the polypeptide Samples of fermented soymilk were collected at pH 5.8 and 5.5
distribution of the soybeans, the beans were milled with a grain and analysed by SDS–PAGE to determine if there was any protein
mill (IKAÒ Works Inc., Wilmington, NC) and the ground flour was hydrolysis occurring during fermentation. Fermented soymilk
analysed using the Dumas combustion method in a LECO FP-528 was analysed for proteolysis at pH values soon after the gel point,
(Leco Corp., St. Joseph, MI) with 6.25 as a conversion factor for % since if proteolysis was occurring at an extent sufficient to alter the
protein from % N (AACC method 46–30.01, 1999). The protein con- gel point, it is expected that proteolysis would have been detect-
centration in soymilk was also measured with Dumas, while solids able by such pH values. The method employed has been previously
were determined by weighing 1 mL soymilk in dry aluminium described others (Keerati-u-rai & Corredig, 2009). The gels were
pans containing dry sand (Fisher Sci., Mississauga, ON, Canada) scanned using a Bio-Rad Gel Doc™ EZ Imager (Bio-Rad, Mississau-
as a dispersing agent. The pans were placed into an IsoTemp forced ga, ON, Canada). The protein composition of fermented soymilk
air oven (Fisher Sci., Mississauga, ON, Canada) at 105 °C for 24 h. was compared to that of unfermented soymilk and of unfermented
Particle size distribution of the soymilk was determined using laser soymilk incubated at 40 °C for 2.5 h.
1718 A. Grygorczyk, M. Corredig / Food Chemistry 141 (2013) 1716–1721
2.5. Gel microstructure Woodrow, & Corredig, 2008) that after centrifugation at 8000g,
soymilk exhibited a monomodal distribution with an average par-
Images of the gel microstructure were taken using an inverted ticle size of 0.2 lm. The distribution was mostly due to soymilk
confocal scanning laser microscope (Leica TCS SP2, model Leica protein particles. In the present work centrifugation was not car-
DM IRE2, Leica Microsystems CMS GmbH, Mannheim, Germany) ried out, hence fat globules were present, together with larger pro-
with an Ar/Kr visible light laser and 63 (oil) objective. Resolution tein particles and some cell wall debris. It was indeed
of the acquired digital images was 1024 1024 pixels. A fluores- demonstrated that the centrifugation step decreases the amount
cence dye (rhodamine B) (Fisher Sci., Fairlawn, NJ) (excitation of total solids and causes creaming of the fat globules in soymilk
and emission wavelengths 543 and 625 nm, respectively) was used without decreasing significantly the amount of protein (Malaki
for staining the protein. Twenty microlitre of rhodamine B (0.2% w/ Nik et al., 2008).
v in milli-Q water) were added to 5 mL of sample for staining. Two Acidification was conducted with addition of 0.6% GDL or the
drops of sample were placed into grooves of a concave microscope addition of lactic acid bacteria. The differences in the pH profiles
slide and a cover slip was placed over the top and sealed. Slides between the treatments are shown in Fig. 2. Similar data have been
were incubated at 40 °C until pH 5.1 was reached, and then cooled shown previously, for example, in milk matrices (Amice-Queme-
in a refrigerator for at least 30 min at 4 °C before viewing under the neur, Haluk, Hardy, & Kravtchenko, 1995; Lucey et al., 1998). The
microscope. lactic acid bacteria culture was able to acidify soymilk to pH 5.1
in about 240 min. The decrease in pH caused by bacterial acidifi-
2.6. Statistical analysis cation began slowly, most probably due to an adapting lag phase
of the bacteria, which lasted about 100 min. After this initial phase,
Measurements were carried out in triplicate and treatments where the pH was constant at about 6.6, the pH started to decrease.
were tested for equal variances then analysed using an unpaired After approximately 4 h, a pH of 5.1 was reached. In contrast with
student’s t-test assuming equal variances, using the using the data the slow acidification by lactic cultures, when 0.6% GDL was added
analysis function in Microsoft Office Excel 2007. the pH drop was very rapid due to the fast hydrolysis of GDL. After
about 50 min, acidification slowed down as GDL began to approach
equilibrium and the soymilk reached pH 5.1 after approximately
3. Results and discussion 2 h. This value of pH was therefore used to compare the final gels.
A comparison between the two modes of acidification allows the
In the present study the soymilk was prepared using common determination of possible differences in the acid-induced gelation
household practises, using only food-grade ingredients. The soy- behaviour of the soymilk particles with varying rates of
beans, purchased from a local grocery store contained 36.8 ± 0.2% acidification.
(n = 3) protein and 15.32 ± 0.01% fat (n = 3) (dry basis), and had a The development of the gel structure during acidification was
ratio between glycinin and b-conglycinin polypeptides of approxi- followed using rheology and a summary of the measured rheolog-
mately 1.7, as determined by SDS–PAGE and densitometry.
The soymilk obtained had a total solids content of 8.55 ± 0.09%,
4.00 ± 0.04% protein and 1.0 ± 0.3% fat. Mineral analysis revealed 7.0
that the soymilk contained the following amounts of minerals
6.8
per kilogramme of soymilk: 250 mg calcium, 330 mg magnesium,
6.6
630 mg phosphorus, 2100 mg potassium, 87 mg sodium and
360 mg sulfur. 6.4
The particle size distribution of the soymilk (Fig. 1) was mea- 6.2
sured using integrated light scattering. The soymilk particles were
pH
6.0
characterised by a bimodal distribution, with two populations at
5.8
approximately 0.2 and 10 lm. These two populations represented
mostly soymilk protein particles and fat globules. Incubation of the 5.6
samples with 1% SDS did not affect the size distribution (data not 5.4
shown). It has been previously reported (Malaki Nik, Tosh, Poysa, 5.2
5.0
0 50 100 150 200 250
4 Time (min)
2
Table 1
Rheological parameters measured during acidification and at a final pH of 5.1 for
soymilk acidified with GDL or with lactic acid bacteria. Gelation pH (measured as tan
1 d = 1), values at tan d at plateau; value of G0 at pH 5.1, slope of frequency sweep
dependence of G0 ; strain at yield. Values are the average of three independent
experiments, with standard deviation. Within a row, different letters indicate
0 statistical difference at p < 0.05.
Tan δ
0.6 used, the soymilk approaches the isoelectric point of soymilk very
200 slowly, allowing more time for proteins to rearrange and interact
0.4 with each other, resulting in a higher gelation point. When GDL
100 is used, the isoelectric point of soy proteins is surpassed very rap-
0.2 idly so it is possible that the pH must drop further before proteins
have time to interact sufficiently to generate a gel structure.
0
0.0 Indeed, the complex composition of the soymilk protein may cause
6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 heterogeneities in the structure of the soymilk particles, and with
pH more time, particles may be able to orient themselves more opti-
mally for short-range attractions. It has been previously shown
Fig. 3. Development of the elastic modulus (G0 ) (filled symbols) and tan d (empty
(Ringgenberg et al., 2013) that the basic subunits of glycinin and
symbols) during acidification of soymilk with bacterial cultures (circles) or GDL
(triangles). The results are representative of three replicates. For statistical analysis b subunits of b-conglycinin begin to aggregate above the gel point,
see Table 1. probably because of their elevated isoelectric points (pH 6.8–8.5
and 5.7–6.0, respectively) (Staswick, Hermodson, & Nielsen,
ical parameters is shown in Table 1. Fig. 3 illustrates the changes in 1981; Thanh & Shibasaki, 1977). As these protein subunits ap-
the elastic modulus (G0 ) and tan d (where d is the phase angle) as a proach a net neutral charge and aggregate, the other subunits of
function of pH for soymilk acidified with GDL or with bacterial cul- soy proteins, which are covalently linked to basic glycinin subunits
tures. The environmental pH is critical to soy protein stability: dur- and b subunits of b-conglycinin, may also approach each other.
ing acidification, protons gradually neutralise surface charges. When these subunits are in closer proximity to each other, short-
When the net charge approaches zero, soymilk particles destabi- range interactions may begin to take place and contribute to the
lise, aggregate and form a particulate gel network (Kohyama development of a gel network. However, until the pH is reduced,
et al., 1995). The gelation pH was defined as the point where tan some repulsion still exists as these subunits have lower isoelectric
d = 1. points (Staswick et al., 1981; Thanh & Shibasaki, 1977). With the
In both cases, the initial value of the elastic modulus (G0 ) ample time provided by bacterial acidification for proteins to rear-
remained very low and constant until the point of gelation, which range into optimal conformations for interaction, the initial aggre-
was earlier in the case of lactic acid bacteria fermentation. The gation of basic subunits of glycinin and b-subunits of b-conglycinin
sudden increase in G0 , which occurs around the isoelectric point may be sufficient to make a network before the remaining protein
of soy proteins, is in full agreement with earlier reports, indicating subunits are incorporated as well. Fig. 3 also shows that during
that the gelation of the soymilk particles is a result of short range structure development, there was a statistically significant differ-
interactions, as gelation occurs when protein particles have ence in the slope of the increase of G0 as a function of pH. Although
reduced repulsive charges and can be in close proximity to each bacterial acidification results in a higher gelation pH, the presence
other (Malaki Nik et al., 2008; Ringgenberg et al., 2013). However, still of a number of proteins with repulsive charges would prevent
there was a significant difference in the pH of gelation of the a rapid development of the gel network.
soymilk particles depending on the mode of acidification (Table As previously mentioned, although the gelation occurred earlier
1). The gelation pH for soymilk acidified with lactic acid bacteria for soymilk acidified with bacteria, and the structure development
was 6.29 ± 0.05. After this pH, the G0 began to increase and contin- also appeared earlier, the small deformation properties of the gel at
ued to increase in a smooth upward curve until the end of the pH 5.1 did not differ. Fig. 4 illustrates the frequency dependence of
gelation experiment (which was arbitrarily set at pH 5.1). The aver- soymilk acid gels produced using a bacterial culture and using GDL.
age final G0 at pH 5.1 was 467 ± 62 Pa. On the other hand, when The curves using both modes of acidification are parallel to each
soymilk was acidified using GDL the gel point was pH 5.9 ± 0.04, other and rise with increasing frequency. The log/log slopes of fre-
in agreement with previous researchers (Malaki Nik et al., 2008,
2011; Ringgenberg et al., 2013). The average final G0 of soymilk
acidified with GDL was 412 ± 72 Pa. This was not significantly dif- 1000
ferent from the final G0 when bacterial acidification was used. Both
gels also showed a similar final tan d value. Although the values of
G0 and tan d for the two gels at pH 5.1 were similar, there seemed
to be a quicker gel development with pH for the gels prepared with
GDL. The values of tan d also reached a similar final value, regard-
G' (Pa)
Fig. 5. Confocal microscopy of the soymilk gels prepared with bacterial culture (A) or GDL (B). Bar size 50 lm.
quency dependence on G0 for the GDL-induced soymilk gel and the Acknowledgements
bacteria-induced soymilk gel (0.073 and 0.072, respectively) were
not statistically different (Table 1). The positive slope suggested This research was supported by the National Science and
that the gels are particulate in nature, as has been previously Engineering Council of Canada and the Hannam Soybean Utiliza-
reported for soymilk acid gels (Malaki Nik et al., 2011). The average tion Fund. The authors would like to thank Danisco Canada
yield strain of soymilk gels formed using a bacterial culture was (Scarborough, ON, Canada) for providing the bacterial culture.
0.113 ± 0.063 and the same value for the gel made by GDL acidifi-
cation was 0.128 ± 0.046. These values were also not found to be
significantly different, again suggesting that the final gel formed
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