CHAPTER 3

Investigation of the efficiency of

encapsulation of probiotics in an
interpolymer complex in supercritical
carbon dioxide using scanning electron
microscopy

Published: F. S. Moolman, P. W. Labuschagne, M.S. Thantsha, T. L. van der Merwe, H.

Rolfes and T. E. Cloete. (2006). Encapsulating probiotics with an interpolymer complex
in supercritical carbon dioxide. South African Journal of Science 102, 349-354.

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3.1 ABSTRACT

Probiotics, beneficial microorganisms, must be available in certain numbers for them to

produce their beneficial effects. The problem of low viability and stability of probiotics
is well known worldwide. Microencapsulation, a technique for coating or protecting
sensitive actives from detrimental environmental factors, has been used by various
researchers in an attempt to solve this problem. However the methods used for
microencapsulation still generally involve exposure of probiotics to water or other
solvents, heat, oxygen, etc. during the encapsulation process, which compromises the
stability of probiotic cultures. A novel method of encapsulation using formation of an
interpolymer complex in supercritical carbon dioxide was developed. This study reports
on the use of scanning electron microscopy (SEM) to investigate the efficiency of the
newly developed encapsulation method in terms of the effect of the encapsulation process
on the cell’s morphology. The effect of the encapsulation process on stability of the
bacterial cells was also investigated. SEM images indicated liquefaction of both
polymers (poly (vinyl pyrrolidone) (PVP) and poly (vinyl acetate-co-crotonic acid)) (VA-
CA) in scCO2. Complete encapsulation of Bifidobacterium lactis cells was achieved,
indicated by absence of bacterial surfaces on the encapsulated particles. Encapsulation of
B. lactis within the interpolymer complex produced smooth textured particles that were
less porous when compared to non-encapsulated freeze-dried bacteria powder. Pores
may allow contact between cells and unfavourable environmental factors. No visual
morphological changes to B. lactis cells were observed due to the encapsulation process.
Survival of non-encapsulated cells and cells that were exposed to the encapsulation
process was similar. Thus, the encapsulation process did not negatively affect stability
and viability of bacterial cells. The successful encapsulation of the bacterial cells within
the interpolymer complex, the absence of changes to cell morphology and the use of
FDA-approved polymers give the technology potential for application in the food and
pharmaceutical industries.

Keywords: interpolymer complex; supercritical carbon dioxide; encapsulation; probiotics,

Bifidobacterium lactis, poly (vinylpyrrolidone), poly (vinyl acetate co-crotonic acid)

73
3.2 INTRODUCTION

The World Health Organization and Food and Agriculture Organization of the United
Nations (FAO/WHO, 2001) define probiotics as “live microorganisms which when
administered in adequate amounts confer a health benefit on the host” (Leahy et al.,
2005). High numbers of viable probiotic cultures are necessary for production of
beneficial health effects (Salminen et al., 1996; Holzapfel et al., 1998; MacFarlane and
Cummings, 1999; Miguel, 2001; Teitelbaum and Walker, 2002; Talwalkar et al., 2004).
Maintanance of viability of the cultures during processing and storage presents a serious
technological and marketing challenge for incorporation of these cultures in functional
foods by industries.

It is difficult and sometimes even impossible for manufacturers to back up claims on their
product labels due to unstable shelf lives of probiotic cultures (Siuta-Cruse and Goulet,
2001). Several market surveys reported a decline in the counts of Lactobacillus
acidophilus and Bifidobacterium spp. during the shelf life of commercial products
containing probiotics, with cell numbers significantly lower than the recommended levels
at the end of shelf life (Micanel et al., 1997; Vinderola et al., 2000; Elliot and Teversham,
2004; Huff, 2004). The survey done in this project on fermented probiotic products
available on South African retail store shelves indicated that there was a problem of
survival of probiotics, especially bifidobacteria, in products.

Over the years, microencapsulation, the process whereby the core material is captured in
a shell or coating for controlled release, has been used. Through microencapsulation,
cells can survive processes such as freezing and freeze-drying better, as well as be
protected from attack by bacteriophages (Krasaekoopt et al., 2003). Protection of
probiotics by encapsulation in hydrocolloid beads has been investigated for improving
their ability in food products and the intestinal tract. Researchers favour the use of
extrusion and emulsion for encapsulating microbial cells (Krasaekoopt et al., 2003). The
disadvantage of using emulsions is that production of large quantities of beads and

74
washing them free of oil is difficult (Stormo and Crawford, 1992). It is also difficult to
produce gel beads at a large scale due to a number of reasons as discussed in Section
1.7.2.2 (Krasaekoopt et al., 2003; Picot and Lacroix, 2004).

Several techniques such as spray drying and fluidized bed drying are used for
encapsulating the cultures and converting them into a concentrated form. One of the
disadvantages of these techniques is that the bacteria are completely released in the
product. Thus the cells are not protected from the product environment and during
passage through the stomach or intestinal tract (Krasaekoopt et al., 2003). Toxic organic
solvents accumulate in microbial cells and kill them through destruction of the functional
properties needed for their survival (Sardessai and Bhosle, 2002; Matsumoto et al., 2004).

Organic solvents are not only toxic to cells, they are expensive as well. Negative effects
of environmental factors such as moisture, temperature and oxygen on probiotic cultures
must be minimized. Medical and food industries require ultra pure products. All these
reasons indicate that new peocessing techniques that can fulfil all these requirements
must be developed (Vasishtha, 2003). An encapsulation technology that would overcome
the problems posed by current technologies, enabling protection and preservation of
sensitive substances, improvement of their viability, effectiveness and shelf lives should
therefore be developed.

The challenges in developing commercially viable encapsulated products depend on:

• Selection of appropriate shell formulation from FDA-approved GRAS (generally
recognized as safe) materials
• Selection of the most appropriate process to provide the desired morphology
• Stability and release mechanism
• Economic feasibility of large scale production, including capital, operating and
other miscellaneous expenses, such as the transportation cost, regulatory cost and
downtime losses.

75
Supercritical fluids (SCFs) are fluids heated to temperatures and pressures above their
critical temperature and pressure. They are able to solubilize compounds and can
penetrate low porosity materials (Demirbas, 2001). SCFs have gas-like diffusivity and
liquid-like densities (Reverchon and Porta, 2001). Though they were originally used for
extraction, experience accumulated in recent years on their use and processes indicated
the possibility to explore and envision their use beyond the common practice of
extraction (Reverchon and Porta, 2001; Sarrade et al., 2003). Supercritical fluid
technologies can also be applied in making new innovative products (Sihvonen et al.,
1999; Reverchon and Porta, 2001). Encapsulation of drugs for release at specific sites in
the human body is one of the new areas for application of supercritical fluid technology
(Sihvonen et al., 1999).

Supercritical carbon dioxide (scCO2), has received increasing attention due to its cost
effectiveness and environmental friendliness (Bae et al., 2004; Novik et al., 2006). The
relatively low critical parameters (Tc = 31.1 ºC and Pc = 73.8 bar1) of scCO2 lends it
towards processing of pharmaceuticals and other materials that are sensitive to
temperature, solvents, oxygen, water, etc. such as proteins, labile drugs and bacteria
(Reverchon and Porta, 2001; Bae et al., 2004). An additional advantage of using SCFs as
solvents, particularly in pharmaceutical applications is that there is no residual solvent in
the final product (Corrigan and Crean, 2002). Typical applications of scCO2 in
biotechnology include micronization of drugs and encapsulation of sensitive actives for
controlled release of the immobilized material (Jung and Perrut, 2000; Fages et al., 2004;
Ginty et al., 2005; Yeo and Kiran, 2005).

Most polymers are not sufficiently soluble in or compatible with scCO2 and can thus not
be processed using it as a solvent or plasticiser. Different approaches can be used to
overcome the problem of insolubility and incompatibility between polymers and scCO2,
though they are typically not allowed in food and pharmaceuticals. The different
approaches used and reasons for their limitation in these industries are given in Table 3.1.

76
Polymers with complementary sites/ molecular groups can interact with each other in
solution to form physical networks by interpolymer complexation (Tsuchida, 1994;
Henke et al., 2005). Interpolymer complex assemblies form through any of four
fundamental attractive interactions, namely electrostatic attraction, hydrogen bonding,
hydrophobic interaction and Van der Waals forces (Henke et al., 2005). It has been
shown that hydrogen bonding (Tilly et al., 1994) and dipole-dipole interactions (Ekart et
al., 1993) can occur in scCO2. scCO2 has largely been used in the food industry for
extraction of labile food components and in pharmaceutical industries for extraction and
purification of vitamins. Recently Novik et al. (2006) reported the use of scCO2 in the
probiotics field for extraction of glycolipids from Bifidobacterium adolescentis 94 BIM.
However, the formation of interpolymer complexes in scCO2 and its application in
encapsulation of probiotic bacterial cells was to our knowledge, reported for the first time
by Moolman et al. (2005).

The main objectives of this study were therefore to investigate the efficacy of this novel
encapsulation technique based on interpolymer complexation in scCO2 using SEM, the
effect of encapsulation on cell morphology and to determine the effect of the
encapsulation process on stability of bacteria using conventional plating techniques.

77
Table 3.1: Approaches to overcoming incompatibility between scCO2 and most
polymers (from Moolman et al, 2006)
Approach Elaboration Limitations Reference
Changing Incorporation of “CO2-philic” FDA approval needed for Sarbu et al., 2000
polymer functional groups in new polymers new polymers
design
Surfactants Addition of CO2 soluble surfactants FDA approval needed for Hoefling et al.,
surfactants 1993; McClain et
al., 1996; Yazdi et
al., 1996
Cosolvents Addition of cosolvents , e.g. methanol Reintroduces requirement Ekart et al., 1993;
or ethanol, to increase the solvation for use of a solvent-many Kazarian et al.,
power of scCO2 actives are sensitive to 1998; Mishima et
solvents al., 2001; Corrigan
and Crean, 2002
nd nd
Mixtures of The use of 2 SCF to enhance No obvious 2 SCF with
SCFs polymer processability desired combination of
properties (low/no
toxicity, low critical
temperature and pressure,
low cost, etc.)
Gas anti- Use of scCO2 as an anti-solvent to Reintroduces requirement Subramanian et al.,
solvent extract the solvent from a sprayed for use of a solvent-many 1999
technique polymer solution and thus precipitate actives are sensitive to
the polymer solvents
Use of low Polymers are more amenable to scCO2 These polymers generally Rindfleisch et al.,
molar mass processing have low mechanical 1996
and low integrity and/or barrier
polarity properties
polymers
Use of Fats, waxes and oils are generally Limited flexibility with Benoit et al., 2000
fats/waxes for soluble in scCO2 regards to properties
encapsulation

78
3.3 MATERIALS AND METHODS

3.3.1 Bacterial cultures

Bifidobacterium lactis Bb-12 and Bifidobacterium longum Bb-46 were obtained as DVS
sachets from CHR- Hansen.

3.3.2 Encapsulation of bacteria

Bifidobacterium cells were encapsulated using a Particles from Gas-Saturated Solution

(PGSS) reactor (Fig. 3.1). All equipment was wiped with 70 % ethanol using a paper
towel, and allowed to dry before contact with the materials. 2 g of PVP (Kollidon 12PF,
mass-average molar mass 2 000 – 3 000 g/mol, BASF) was dried for 5 h at 80 ºC and 60
mbar (absolute) in a vacuum oven (Model VO65, Vismara) and immediately placed in a
dessicator to prevent moisture absorption. A sealed packet of either B. longum Bb-46
(Chr. Hansen) or B. lactis Bb-12 (Chr. Hansen) was removed from storage at -12 ºC and
allowed to warm to room temperature while sealed. 2 g of the bacteria was then ground to
a powder passing through a 150 µm sieve using a coffee grinder (Model CG100,
Kenwood). 6 g of VA-CA (Vinnapas C305, mass-average molar mass 45 000 g/mol,
Wacker) was then added to the bacteria (together with any additives (e.g. glyceryl
monostearate – Croda Chemicals, in reactions were additives were included) and the
dried PVP. The blend was then ground and mixed for 1 min. The powder blend was then
immediately transferred to the pre-heated 1 reaction chamber. The chamber was then
sealed and flushed and pressurized with sterile filtered CO2 (99.995% purity, Air
Products) up to a pressure of 300 bar, with the temperature controlled at 40 ºC. The
material was left to equilibrate for 2 h with intermittent stirring, after which the liquefied
product was sprayed through a 500 µm capillary with length 50 mm, into a 10
expansion chamber that was pressure-controlled at 15 bar (gauge). Fig. 3.2 is a
simplified flow diagram showing steps occurring in the encapsulation process. A clear
description of the encapsulation technology is outlined in Moolman et al. (2006).

79
Figure 3.1: A 1 PGSS reactor (Separex Equipments, France) used for
encapsulation of bacteria, situated at the Polymers and Ceramics centre, CSIR,
Brummeria, PTA. A= CO2 supply, B= Pump, C= CO2 preheater, D= Mixing
chamber, E= Product chamber, F= Control panel

80
Figure 3.2: A simplified process flow diagram of the PGSS (Particles from Gas-
Saturated Solution) system used to produce encapsulated probiotics

3.3.3 Scanning electron microscopy (SEM)

SEM was used to verify encapsulation of B. lactis cells into the polymer and release of
the cells from the polymer into solution during subsequent suspension of the encapsulated
material. The freeze-dried and encapsulated bifidobacteria were suspended in ¼ strength
Ringer’s solution. The suspended cells were filtered out using a 0.2 m Millipore filter
membrane. The cells were fixed to the 0.2 m membrane using 2.5 % gluteraldehyde for
30 min. The fixed cells were then washed 3 x 15 min in 0.15 M-phosphate buffer. Then
dehydration of the sample was done in an increasing series of ethanol as follows: 50 % (1
x 15 min), 70 % (1 x 15 min), 90 % (1 x 15 min) and 100 % (3 x 15 min). The filter

81
membrane was then dried in a critical point dryer for 24 h, mounted on stainless steel
studs and then coated with gold plasma. The freeze-dried and encapsulated powder were
put on a sticky tape on the studs and directly coated with gold plasma without undergoing
any treatments for SEM. The samples coated with gold were then examined using JEOL
840 scanning electron microscope.

3.3.4 Bacterial counts

1 g of Bifidobacteria was suspended in 9 m of sterile ¼ strength Ringers solution (pH

7). A series of dilutions up to 10-10 was prepared from this suspension. 0.1 m of
appropriate dilutions was pour plated onto De Man, Rogosa and Sharpe (MRS) agar
(Merck, Pty.(Ltd)), supplemented with 0.05 % cysteine hydrochloride. Each dilution was
plated out in triplicate. The plates were incubated anaerobically in anaerobic jars with
Anaerocult A gaspaks (Merck Pty (Ltd.), at 37 oC for 72 h. Anaerobisis inside the jars
was indicated by inclusion of Anaerocult C test strips (Merck, Pty (Ltd)). The numbers
of colonies grown were counted and from these the numbers of viable cells were
calculated (cfu/g). Reported values are averages of the three replicates.

3.4 RESULTS AND DISCUSSION

3.4.1 Liquefaction of polymers in scCO2

SEM was used to examine whether the developed SCF encapsulation method was
efficient and whether the polymers used were liquefied during exposure to scCO2. SEM
images of PVP and VA-CA before and after exposure to scCO2 are shown (Fig. 3.3).
These images indicated that liquefaction of both polymers occurred during exposure to
scCO2 (Fig. 3.3). Images before exposure to scCO2 showed polymers as individual
granules (separate loose particles with individual particles/ granule’s three dimensional
structure visible showing that the particles were separate) (Fig. 3.3 A, C) while those
after exposure appeared as a monolithic foam (Fig. 3.3 B, D). No individual particles
similar to those observed before exposure to scCO2 were present. The continuous

82
appearance (compact layer) was the result of liquefaction of the polymers by the
dissolution of scCO2 in the polymers. Dissolution of scCO2 in polymers is known to
lower glass transition temperature (Tg) and facilitates formation of a smooth morphology
(Yue et al., 2004).

A B

C D

Figure 3.3: Comparison of PVP before (A) and after (B) and VA-CA before (C) and
after (D) scCO2 exposure

3.4.2. Interpolymer complexation and bacterial encapsulation

Encapsulation of B. lactis Bb-12 cells by the interpolymer complex formed between PVP
and VA-CA was achieved (Fig. 3.4 B). PVP is a water soluble inert polyamide polymer
with complexing properties and has been used in pharmaceutical products (Kumar et al.,
1999). It has carbonyl groups while VA-CA has carboxylic acid groups (Raveendran et
al., 2005). Therefore it was expected that these two groups would interact with each
other through hydrogen bonding and form a complex between the two polymers. Such an

83
interpolymer complex was indeed formed through the formation of hydrogen bonds
between the carboxylic acid groups and carbonyl groups of VA-CA and PVP,
respectively (Moolman et al., 2006). Similar results were observed by other researchers
(Raveendran et al., 2005). Surface characteristics or appearance of encapsulated material
and freeze-dried bacterial powder were different. Even though the freeze-dried cells
were clumped together, the rod-shaped individual bacterial cells forming the clumps
could be seen (Fig. 3.4 A). It could be observed from the images that there was no layer
around or covering the bifidobacteria cells to offer protection. Thus the non-encapsulated
freeze-dried bacteria had no shield to protect them should they be exposed to detrimental
factors during storage or after ingestion.

On the other hand, no bacterial cells were visible on surfaces of the encapsulated
material. The encapsulated bacteria were therefore completely enclosed within an
interpolymer complex formed. The ability of polymers to interact with each other in
solution through secondary binding forces such as hydrogen bonds, dispersion forces and
hydrophobic interactions, to form intermacromolecular complexes is well known (Henke
et al., 2005). The interpolymer complex surrounding the bacteria serves as a potential
barrier against detrimental environmental factors to which the probiotic bacteria are
normally exposed, such as oxygen during storage and gastric acid during transit through
the gastrointestinal tract.

84
 A B

C D

E F

Figure 3.4: SEM images of Bifidobacterium longum Bb-46 cells: A and C: Non-
encapsulated (powder), B: scCO2 interpolymer complex encapsulated (powder), D:
Non-encapsulated (suspension), E and F: scCO2 interpolymer complex encapsulated
(suspension)

85
Water insoluble dry capsules for incorporation of bifidobacteria in food products should,
according to Doleyres and Lacroix (2005), have a particle size of <100 m for stability,
easier handling and storage of cultures as well as limited effects on the product texture.
The size of particles produced using the method described in this chapter can be
controlled through the use of additives such as glyceryl monostearate. Addition of
glyceryl monostearate reduced the particle size by more than an order of magnitude
(results not shown).

3.4.3 Appearance of non-encapsulated and encapsulated cells upon suspension

Suspension of encapsulated powder in sterile distilled water (pH 6.8) released the
encapsulated bifidobacteria cells from the interpolymer complex into the
solution/suspension. PVP is water soluble (Kumar et al., 1999) while VA-CA is
insoluble. Though VA-CA is insoluble in water, higher pH results in dissociation of the
crotonic acid groups, leading to increased compatibility with water (Moolman1, Personal
communication). This then causes VA-CA to swell, causing the release of the bacteria
enclosed within the interpolymer complex. The residues of the disrupted polymer were
visible between the bacterial cells and on the mounting surface. When the released
suspended cells were viewed under SEM no visible differences between the
bifidobacteria cells released from the interpolymer complex (Fig. 3.4 E) and the freeze-
dried non-encapsulated bacteria in suspension (Fig. 3.4 D) were observed. This indicated
that the encapsulation process did not produce any noticeable damage or morphological
changes to the bacteria. Thus it seemed that the encapsulation process did not negatively
affect the enclosed cells, but this can not be concluded from the SEM results alone.
The numbers of cells captured from 1 ml of the suspension were high for both non-
encapsulated and encapsulated bacteria. The method therefore allowed encapsulation of
high numbers of cells which is an advantage when looking at the envisaged application of
the method in probiotic foods whereby high levels of viable bacteria are required for
production of beneficial effects.

1
Moolman F. S. Polymers, Ceramics and Composites, Materials Science and Manufacturing, CSIR, P. O.
Box 395, Pretoria, 0001, South Africa

86
3.4.4 Viability of Bifidobacterium longum Bb-46 cells after scCO2 processing

SEM images were able to show satisfactory encapsulation of bacteria and an unchanged
physical appearance of cells upon suspension, but could not tell whether the cells were
alive or dead. Plate counting technique was applied to fulfil this crucial purpose. Fig. 3.5
shows counts of bacteria over six weeks. Numbers of viable bacteria for both
unprocessed and scCO2 processed cells decreased over the storage time.

1.E+11
Bacterial counts (cfu/g)

1.E+09

1.E+07 Unprocessed Bifidobacterium longum Bb-46 cells

SCF-processed Bifidobacterium longum Bb-46 cells

1.E+05
0 1 2 3 4 5 6 7
Time (weeks)

Figure 3.5: Survival of freeze-dried bacteria and freeze-dried bacteria that went
through the encapsulation process during storage

The reduction in numbers of viable bacteria over time for scCO2 processed and
unprocessed bacteria were similar (Fig. 3.5). The counts of both scCO2 processed and
unprocessed cells were around 2 x 1011 cfu/g at the beginning of the trial and reduced to 3
x 108 cfu/g after 6 weeks of storage at 30 oC (Fig. 3.5). There was no significant
difference in the numbers of live bacteria for both samples over 6 weeks. The results
indicated that not only did the exposure to scCO2 not produce noticeable effects to the
morphology of the processed bacteria, but the stability and therefore viability of the cells

87
was also not negatively affected. Probiotic bacteria encapsulated using the described
method should thus remain viable within the interpolymer complex after encapsulation
and produce beneficial effects upon release at the desired site.

3.5 CONCLUSIONS

Satisfactory liquefaction of VA-CA and PVP upon exposure to supercritical carbon

dioxide was achieved. Successful encapsulation was indicated by the absence of
bifidobacteria on interpolymer complex surfaces and by the release of high numbers of
bifidobacteria from the interpolymer complex upon suspension. No undesirable effects
such as morphological changes or reduced cell stability occurred as a result of the
encapsulation process. The results therefore indicate for the first time, the potential of
interpolymer complexation in scCO2 for application in food and pharmaceutical
industries for encapsulation of sensitive probiotic bacteria in order to protect the cells
from detrimental factors leading to unwanted reduction in viability.

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