Advancements in microbial polysaccharides research for frozen foods and
microencapsulation of probiotics
Y. Martin Lo, Patrick D. Williams, and Pavan Kumar Soma
Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, USA
(e-mail: [email protected])
ABSTRACT
Conventionally used in the food industry as stabilizing, thickening, gelling, and suspending or dispersing
agents, microbial polysaccharides such as xanthan gum are known to improve the texture of certain frozen
products. Another polysaccharide that has received significant attention in recent years is chitosan, a natural
biopolymer derived from chitin. In the wake of growing interest in finding ideal encapsulating agents for
probiotics, microbial polysaccharides interacting with chitosan have been investigated. Scattered research
could be found on the effect of each individual polysaccharide; however, there remains a void in the literature
to closely compare the characteristics of microbial polysaccharides for these applications, especially when
more than one biopolymer is employed. A good understanding of tools capable of elucidating the underlying
mechanisms involved is essential in ushering further development of their applications. Therefore, this study
aims at establishing the selection criteria of microbial polysaccharides based on their rheological properties,
resistance to harsh conditions, and ability to improve sensory quality. A variety of critical tools is also
carefully examined with respect to the attainable information crucial to frozen food and microencapsulation
applications.
Keywords: microbial polysaccharide; xanthan; curdlan; chitosan; hydrogel.
INTRODUCTION
Microbial polysaccharides such as xanthan gum have conventionally been used in the food industry as
stabilizing, thickening, gelling, and suspending or dispersing agents [1]. In the wake of growing interest in
developing quality frozen products and those containing live probiotics, there are respective needs to find
effective mechanisms to eliminate syneresis in frozen products, as well as to identify ideal encapsulating
agents for controlled release of probiotics to endure harsh processing conditions and acidity in the digestive
system. Scattered research could be found on some applications of individual polysaccharides; however,
there remains a void in the literature to closely investigate the characteristics of microbial polysaccharides for
frozen food and microencapsulation applications, especially when more than one biopolymer is employed. In
the present article, two unique hydrogel complex systems containing xanthan gum in conjunction with a
secondary biopolymer are reported. The first system was able to control moisture migration and eliminate
syneresis in frozen foods, whereas the second system successfully protected probiotics against strong acidity
in gastric juice and released the cells by swelling upon contact with bile salt.
MATERIALS & METHODS
Xanthan gum was provided by TIC Gums (Belcamp, MD). Odorless, fine, free-flowing white powder
curdlan containing a minimum of 90% β-D-glucan and with a maximum of 10% water was provided by
Takeda USA (Orangeburg, NY). Chitosan at 85% deacetylation, including low molecular weight (LMW, 20200 cP), medium molecular weight (MMW, 200-800 cP), and high molecular weight (HMW, 1200-1600 cP),
was employed. The hydrogel complexes of xanthan-curdlan (XCu) and xanthan-chitosan (XCh) were
prepared respectively for freeze-thaw and microencapsulation evaluations.
For XCu, the polymers were gradually added to continuously stirred water until well hydrated, then heated to
90°C under reflux using a water bath before cooled to room temperature and stored at 4°C for 24 hrs. To
complete a freeze-thaw cycle (FTC), the samples were placed at -20°C for 18 hrs, followed by 6 hrs of
thawing at room temperature [2]. Syneresis of the hydrogel complexes after each FTC was determined by
centrifuging the samples at 2200 rpm (707 × g) for 20 min in a Beckman Model TJ-6 centrifuge. The volume
of exuded water was determined using a laboratory graduated cylinder. A TA.XT2i texture analyzer
(Texture Technologies, Inc., Scarsdale, NY) was employed to determine the gel strength and adhesiveness
profiles using a P/0.5R probe with a 5-kg load cell. All rheological measurements were performed using a
controlled strain AR2000 rheometer (TA Instruments, New Castle, DE). Magnetic Resonance Imaging
(MRI) and Nuclear Magnetic Resonance (NMR) relaxometry experiments were carried out using a 1.03T
NMR spectrometer with 43.8 MHz for 1H-resonance frequency (Aspect Imaging, Hevel Modi'in Industrial
Area, Israel).
The XCh capsules containing probiotic Lactobacillus acidophilus were optimized [3]. Eosin Y, a fluorescent
dye, was employed to assess the effect of external pH on the internal pH of XCh hydrogel. To assess the
changes in internal pH of the hydrogel over time, various xanthan solutions containing 0.01% (w/v) eosin Y
were prepared. Aliquots of this mixture (150 μl) were added to at least 3 wells in a 96-well plate before
adding 100 μl of chitosan solution. After a predetermined complexation time, excess chitosan was removed
by inverting the plate and tapping gently. To get rid of residual chitosan, each well was washed twice with
pH 4.0 HCl solution to minimize swelling and leaching of dye, followed by adding 100 μl HCl solution (pH
2.75). The emission intensity (EI) was measured on a fluorescence microplate reader (FLUOstar, BMG
Labtech, Offenburg, Germany) with the excitation and emission wavelengths set respectively at 485 and 510
nm. The experiments were conducted in triplicates. The effect of complexation time was characterized, and
the release mechanism was determined based on resulting cell viability.
RESULTS & DISCUSSION
For XCu, consistent apparent viscosity (between 2.8 and 3.5 Pa s) was observed over five FTCs at pH 7.
Increasing curdlan concentration decreased the loss modulus, which stayed stable over the 2-5 FTCs. The
heat stability of XCHC was better when xanthan gum was the dominating polymer in the system. All
combinations of XCu did not show any water loss under conditions studied, indicating XCu is capable of
providing freeze-thaw stability without syneresis. Xanthan is known to exhibit ‘gel-like’ behavior, which is
indicated by its frequency sweep; however, the scanning electron micrographs (SEM) did not reveal a threedimensional network; rather, XCu showed two-dimensional layers closely packed. The lack of a threedimensional network in the SEM micrographs was expected due to xanthan’s dynamic modulus dependency
upon angular frequency and low modulus values (Figure 1a).
XCh microcapsules averaging 300-500 µm after freeze-drying were produced under the optimal processing
conditions. The size of the capsules was most dependent on the aspiration rate, whereas agglomeration was
formed at high feed pump rate. Relatively uniform capsules without agglomeration were formed when 0.7%
(w/v) xanthan solution were sprayed into 0.7% (w/v) chitosan at pH 4.7 using the feed pump and aspiration
rates at 3.6 ml/min and 6.6 m3/hr, respectively. The concentration of xanthan was found the most crucial,
whereas chitosan solution properties were negligible, in determining the optimal formulation. However, with
xanthan concentration exceeding 1.25 wt/v % only amorphous beads were formed. Encapsulated cells could
be completely release in 0.1% peptone at pH 8 after 1 h. The XCh gels improved cell viability at pH 2 by
two-log compared to suspending cells. Protective effects were observed at freezing temperature and at 60ºC
(Figure 1b).
(a)
(b)
Figure 1. Scanning electron micrographs of (a) XCu formed by 1% (w/v) xanthan and 1% (w/v) curdlan; and (b) XCh
containing probiotic Lactobacillus acidophilus ATCC 43121 cells.
It was found that adjusting the pH, above or below the original pH of XCu (ca. 5.83), caused a reduction in
dynamic modulus values during frequency sweeps (Figure 2). However, the moduli of XCHC with pH of 2
began to increase after the second FTC and continued to increase through the fifth FTC above control values.
Furthermore, its modulus was significantly larger than solutions with pH of 3 through 8 over all FTCs.
Figure 2. Frequency sweeps of 2.0% XCu with pH values of 2, 3, 4, 5, 6, 7, and 8. The green and red contours represent
the storage (G’) and loss (G”) modulus, respectively, prior to (a), and after the first (b), second (c), third (d), fourth (e),
and fifth (f) freeze-thaw cycles (FTCs).
The effect of xanthan concentration and chitosan molecular weight on the diffusion of acidic media was
investigated over time (Table 1). The EI was found to decrease with time until it reached a lower limit,
followed by a steady period in all cases with no significant difference (P>0.05) between adjoining values.
Decrease in EI was more pronounced in the case of 0.7% xanthan and it took the longest with 1.2% xanthan.
Xanthan at 1.2% concentration reached a constant level of EI in 60 min, whereas 1.0% and 0.7% plateaued at
45 and 30 min, respectively. This could be attributed to the formation of less permeable hydrogel membrane
due to elevated cross-linking density. The higher polymer concentration resulted in higher charge density at
the point of contact between the two polymers, leading to improved cross-linking density. Chitosan
molecular weight did not have a significant effect on EI of eosin Y. The differences in EI at time 0 could be
attributed to the loss of eosin Y during washing, along with time differences in measuring the samples.
The effect of complexation time was characterized using eosin-containing XCh hydrogels formed by 1.2%
xanthan and 0.7% HMW chitosan. The EI of eosin Y decreased with time in all cases with significant
differences between 40, 80 and 120 min curves up to 30 min of diffusion time. After 30 min, there was no
significant difference between 80 and 120 min complexation time. It is noteworthy that complexation time
of 40 min appeared to be the most desirable profile due to the delay in the decrease in EI compared to 80 and
120 min. This may be counterintuitive as longer complexation time leads to formation of thicker
polyeletrolyte hydrogels, which results in lower permeability [4]. However, in the present study, with
increase in complexation time, the hydrogel was found to shrink due to the higher osmotic pressure exerted
by the external chitosan solution, which consequently caused migration of water from the hydrogel. The
significant difference in the EI at time zero among various complexation times could be postulated by the
loss of eosin Y that escaped from the hydrogel through the same mechanism. Once the chitosan was emptied
and the acidic media was introduced, the reversal in osmotic pressure difference would force the acidic media
into the hydrogel, hence decreasing the EI of eosin Y.
Table 1. Variation in EI of eosin Y with changes in xanthan concentration, chitosan molecular weight, and
time (n=3). Means with the same letter are not significantly different at P=0.05 level.
Chitosan
48635ed
33387ij
20047uvtrsq
18331uvtrws
17284uvtws
16542uvtw
16704uvtw
51843bdc
42639gf
25381knlm
17004uvtw
16075vw
15871vw
15178w
54873abc
50974dc
34276i
25295oknlm
20682uptrsq
18852uvtrqsw
17309uvtws
MMW
0
12
30
45
60
90
120
48964ed
35195ih
22551opnrmq
20002uvtrsq
17877uvtws
17306uvtws
16437uvw
50007d
44836ef
26501klm
20652uptrsq
17575uvtws
17270uvtws
16624uvtw
55846ab
51526bdc
35395ih
26426klm
22895opnlmq
19933uvtrsq
18274uvtrws
HMW
0
12
30
45
60
90
52303bdc
35703ih
22602opnrmq
19214uvtrwsq
17568uvtws
17081uvtw
55526ab
45579ef
27298kl
20918optrsq
18755uvtrwsq
17446uvtws
55997a
52331bdc
39250gh
29488kj
24722oklmnq
21555opnrsq
LMW
120
0.7%
Xanthan Concentration 1.0%
1.2%
Time
(min)
0
12
30
45
60
90
120
16308uvw
16378uvw
19275uvtrwsq
CONCLUSION
The unique ability of xanthan gum to form a hydrogel complex structure with curdlan (XCu) reduced
syneresis to an undetectable level and exhibited elasticity closer to that of curdlan than xanthan – while
lacking a three dimensional network, making XCu an ideal candidate to stabilize moisture in products
susceptible to freeze-thaw abuse.
REFERENCES
[1] Lo Y.M., Ziegler R.C., Agrin-Soysal S., Hsu C.H. & Wagner N.J. 2009. Effects of intermolecular interactions and
molecular orientation on the flux behavior of xanthan gum solution during ultrafiltration. Journal of Food Process
Engineering 32(5), 623-644.
[2] Williams P.D., Sadar L.N. & Lo Y.M. 2009. Texture stability of hydrogel complex containing curdlan gum over
multiple freeze-thaw cycles. Journal of Food Processing and Preservation 33(1), 126-139.
[3] Argin-Soysal S., Kofinas P. & Lo Y.M. 2009. Effect of complexation conditions on xanthan-chitosan polyelectrolyte
complex gels. Food Hydrocolloid, 23, 202-209.
[4] Dumitriu S., Chornet E. 1997. Immobilization of xylanase in chitosan-xanthan hydrogels. Biotechnology Progress,
13, 539-545.