1. Art and Diseño

FEMS Microbiology Letters 194 (2001) 33^37
www.fems-microbiology.org
Rapid CE microbial assays for consumer products that contain
active bacteria
Daniel W. Armstrong *, Je¡rey M. Schneiderheinze, John P. Kullman, Lingfeng He
Department of Chemistry, Iowa State University, Ames, IA 50011, USA
Received 7 August 2000 ; received in revised form 16 September 2000; accepted 22 September 2000
Abstract
Recent advances in high efficiency separation methods of bacteria allow their rapid identification and quantitation in some cases. A
specific capillary electrophoresis (CE) technique is used to identify and quantitate Lactobacillus acidophilus in both pill and syrup health
products as well as Bifidobacterium infantis in a powdered formula supplement. Cell viability can be evaluated as well. In some cases, both
the living and dead bacterial cells as well as the molecular excipients can be evaluated in a single run. ß 2001 Federation of European
Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Capillary electrophoresis ; Cell viability ; L. acidophilus; B. infantis
1. Introduction
There are increasing numbers of health aids, supplements and consumer products in which the `active ingredient' is not a compound, but rather a microbe. Many
reports indicate that speci¢c microbes can be bene¢cial
to human health. Often this is due to the microbe's ability
to produce needed or useful substances [1]. In other instances a microbe may degrade or alter unwanted or detrimental substances [2^7]. Also it is known that a healthy
population of benign microorganisms can inhibit or retard
the growth of unwanted or pathological (in human terms)
species [1,2].
Some of the most prevalent examples of products in
which the active ingredient is a bacterium, are pills and
syrups for individuals that are lactose intolerant. It is estimated that approximately 50 million Americans su¡er
from lactose malabsorption, which produces a number
of clinical symptoms [4]. The purity, activity and e¡ectiveness of the available products for treating lactose maldigestion are open to question [2^7]. One of the problems is
that there are no e¡ective assays for the active bacterial
ingredient(s). This is important since it is known that both
the viability and the nature (species and strain) of the
* Corresponding author. Tel. : +1 (515) 294-1394;
Fax: +1 (515) 294-0838; E-mail: [email protected]
bacteria determine whether or not it produces any bene¢cial e¡ects [1,2,5,7].
There are numerous other examples of commercial bacterial preparations for which there are no fast, e¤cient or
sensitive assays. This includes medicinal, food and other
consumer products. Recently it has been demonstrated
that speci¢c capillary electrokinetic techniques can be
used to rapidly separate, identify and quantitate bacteria
and fungi [8^11]. If it is possible to adapt these methods so
that microbes can be assayed in complex consumer products (in much the same way as molecules) it would be
extremely useful. In this communication we report the ¢rst
high e¤ciency microbial assays of tablets (pills) and powder-based commercial products.
2. Materials and methods
2.1. Materials
Tris(hydroxymethyl)aminomethane (TRIS), boric acid,
ethylenediaminetetraacetate (EDTA), and poly(ethylene)
oxide (PEO; Mn = 600 000) were purchased from Aldrich
(Milwaukee, WI, USA). Milk-free Acidophilus distributed
by Schi¡ Products, Inc. (Salt Lake City, UT, USA) and
Spring Valley brand natural Acidophilus manufactured by
Rexall Sundown, Inc. (Boca Raton, FL, USA) are dietary
supplements containing Lactobacillus acidophilus. Solaray
brand BabyLife1 dietary supplement manufactured by
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 5 0 2 - 4
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D.W. Armstrong et al. / FEMS Microbiology Letters 194 (2001) 33^37
Nutraceutical Corp. for Solaray, Inc. (Park City, UT,
USA) contains Bi¢dobacterium infantis. All three dietary
supplements were purchased from a local nutrition store.
A LIVE/DEAD BacLight Bacterial Viability Kit was purchased from Molecular Probes, Inc. (Eugene, OR, USA).
It consists of SYTO 9 green £uorescent nucleic acid stain
and red £uorescent nucleic acid stain, propidium iodide,
which are dissolved separately in DMSO.
2.2. Methods
A stock bu¡er solution containing 4.5 mM TRIS, 4.5
mM boric acid and 0.1 mM EDTA (TBE bu¡er) was
prepared by dissolving appropriate amounts of each reagent in deionized water yielding a bu¡er of pH 8.4.
This bu¡er solution was then diluted 8:1 with deionized
water (diluted TBE bu¡er). A stock polymer solution was
prepared by dissolving 0.2 g of PEO in 40 ml of the diluted TBE bu¡er. This solution was sonicated for 2^3 h at
50³C (Fisher model FS-28, 720 W at 43 kHz) to facilitate
the dissolving process. The mixture was then removed
from the bath and left to completely dissolve by stirring
overnight at room temperature. The CE running bu¡er
was prepared by diluting the stock polymer solution
with the diluted TBE bu¡er to a ¢nal polymer concentration of 0.025% (v/v). All bu¡ers and polymer solutions
were prepared fresh daily. Cultures were grown in-house
from the three dietary supplements. Samples were added
to Nutrient Broth (Difco Laboratories, Franklin Lakes,
NJ, USA) and were grown for 10^24 h at 30³C on a
shaker at 250 rpm.
ware. Fused silica capillary with a 100 Wm i.d. was purchased from Polymicro Technologies, Inc. (Phoenix, AZ,
USA). The column used for the separations was 27 cm in
length (20 cm to window). Prior to each injection, the
column was washed for 0.5 min with water, 1.5 min
with 1 N NaOH, 0.5 min with water, followed by
0.5 min with the running bu¡er. The samples were pressured injected for 10^12 s at 0.5 psi. The separation was
performed at a voltage of 10 kV and a temperature of
25³C with thermostated control. On-line UV detection of
the samples was accomplished at 214 nm. The detector
response results from a combination of light scattering
and absorbance.
2.4. Quantitation of bacteria
Samples were prepared for quantitation by dissolving 1,
1.5, 2, 3 and 4 Schi¡ tablets in 20 ml of the CE running
bu¡er. Samples were then injected into the P/ACE 5000
CE system for 20 s at 0.5 psi. The separation was performed at voltage of 10 kV and a temperature of 25³C.
On-line detection was accomplished at 214 nm. The relative standard deviation (RSD) of the peak areas was calculated to be 2.85% (n = 3).
2.3. Capillary electrophoresis
The pill samples were prepared by dissolving the tablets
in 10 ml of CE running bu¡er in a 40 ml sample vial. The
sample was shaken by hand and allowed to settle for 20^
30 min under the in£uence of gravity. This allowed the
larger insoluble pieces of the pill to settle to the bottom
of the sample vial leaving a cloudy solution above the
solid particles. 2^3 ml of cloudy solution was then taken
as the sample. Visual microscopy (400U) con¢rmed the
presence of bacteria in the samples and also pieces of unidenti¢ed insoluble particles. The sample removed from
the 40 ml vial was then used for CE analysis. Samples
from the bacterial cultures grown from the tablets were
obtained by removing 6^7 ml of liquid culture and pelleting the cells in a centrifuge (Fisher model 228, Pittsburgh,
PA, USA) at 3400 rpm. The supernatant was decanted
and the cells were washed with 1^2 ml of CE running
bu¡er and again pelleted in the centrifuge. After another
wash, the cells were dispersed in 1^2 ml of CE running
bu¡er and used for analysis.
The CE separations were performed on a Beckman P/
ACE 2100 or P/ACE 5000 (Palo Alto, CA, USA) coupled
to a computer equipped with Gold data acquisition soft-
Fig. 1. Electropherograms of (A) cultured cells of B. infantis from Babylife powder, and (B) direct injection analysis of dissolved Babylife powder. See Section 2 for experimental details.
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equipped with a 520 nm bandpass ¢lter and a 663 nm
longpass ¢lter, respectively. Data were collected with
P/ACE system MDQ software.
The ratio of green £uorescence peak area to the red
£uorescence peak area can be correlated to the ratio of
live cells to dead cells in a pill after correcting for spectral
overlap and normalizing the peak areas to known concentrations of live and dead cells. Relatively high concentrations of the dyes are used (compared to biological staining
procedures) in order to maximize staining of the cells and
produce consistent results. The CE-LIF cell viability results were con¢rmed by £ow cytometry using a Beckman
Coulter EPICS XL-MCL instrument.
3. Results and discussion
Speci¢c electrokinetic separation conditions can be engineered so that microbes of the same type elute as single
peaks [8,9]. This should allow the development of rapid
instrumental assays and quantitation for microorganisms.
While high e¤ciency separation-based assays are common
for molecules, ions, etc., nothing comparable has ever
been demonstrated for microorganisms.
Fig. 2. Electropherograms of (A) cultured cells of L. acidophilus from
Schi¡ tablets, and (B) direct injection analysis of dissolved Schi¡ tablets.
See Section 2 for experimental details.
2.5. Viability determination
Samples were prepared by dissolving one Schi¡ tablet in
20 ml CE running bu¡er. Three ml aliquots of cloudy
solution were taken as samples. Approximately 1 Wl of
20 mM propidium iodide and 10 Wl 3.34 WM SYTO 9
solution was added to a 3 ml sample solution. The sample
was incubated in the dark for 30 min. Generally, SYTO 9
stain labels all bacteria in a population ^ those with intact
membranes and those with damaged membranes. In contrast, propidium iodide penetrates only bacteria with damaged membranes, causing a reduction in the SYTO 9 stain
£uorescence when both dyes are present. Thus, with an
appropriate mixture of SYTO 9 and propidium iodide
stains, live bacteria with intact cell membranes stain £uorescent green, whereas dead bacteria with damaged membranes stain £uorescent red.
Viability determination was completed on a Beckman
Coulter P/ACE MDQ capillary electrophoresis system,
equipped with 488 nm laser induced £uorescence (LIF)
detector. The sample was injected for 5^10 s at 0.5 psi.
The separations were performed at 15 kV and a temperature of 25³C. Green and red £uorescent light were monitored simultaneously with the LIF detector, which is
Fig. 3. Electropherograms of (A) cultured cells of L. acidophilus from
Spring Valley tablets, and (B) direct injection analysis of dissolved
Spring Valley tablets. See Section 2 for experimental details.
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D.W. Armstrong et al. / FEMS Microbiology Letters 194 (2001) 33^37
Fig. 4. Plot showing the correlation between peak area and the number of L. acidophilus cells in injected samples from Schi¡ pills. Such standard curves
(y = 0.046^2.51, R2 = 0.995) allow rapid and accurate quantitation of bacteria.
An electropherogram for a culture grown standard of
B. infantis is shown in Fig. 1A. These bacteria are in
certain products (see Section 2) for introduction to the
guts of newborn infants. The powdered formulation is a
relatively simple mixture of maltodextrin and the bacterium B. infantis. The CE electropherogram of this formulation shows that both the bacteria and the maltodextrin
(which travels with the eof) can be identi¢ed and quantitated in the same run (Fig. 1B).
Several formulations contain L. acidophilus as the active
ingredient. An assay of L. acidophilus from a commercial
syrup (Fig. 2B) shows that the L. acidophilus (as identi¢ed
from the standard in Fig. 2A) as well as at least two of the
molecular components of this preparation can be identi¢ed. Note that the conditions of these assays (i.e. the pH,
strength, bu¡er type and polymer concentration of the
running bu¡er) can usually be controlled so that the larger, charged microbes elute latter than most of the excipients.
Finally L. acidophilus was analyzed from a complex
tablet formulation that contained six or more molecular
or colloidal excipients. The electropherograms of the bacteria standard and a solution of the tablet are shown in
Fig. 3A and B respectively. The purpose of this ¢gure is
two-fold. First, it shows that a bacteria can be assayed in
widely di¡erent formulations (i.e. matrices). Second, mi-
gration times can vary slightly (see L. acidophilus in Fig.
3A vs. B) in the presence of certain matrices. Thus an
internal standard or sample spiking should be used to
verify one's results (at least initially). Most often a component or components of the injected sample change the
eof, which then a¡ects the migration of other components
in that run. This same phenomenon was noted recently
when the direct injection of concentrated urine was used
for the CE diagnosis of urinary tract infections [9].
The CE peak area (Fig. 4) can be directly correlated to
the number of cells injected (or the weight% of cells injected). Thus, the direct and rapid quantitation of cells in a
sample or environment can be done. The ability to both
e¤ciently identify and quantitate microorganisms can be
di¤cult or impossible using conventional or classical
methods.
Electropherograms generated using UV detection (Figs.
1^3) allow identi¢cation of a microorganism and quantitation of the total number of cells (Fig. 4). However, they
give no information on cell viability. Cell viability can be
determined in the same CE run by pretreating the cells
with a mixture of SYTO 9 green £uorescent nucleic acid
stain plus red £uorescent propidium iodide stain and using
dual channel, laser induced £uorescence detection (see Section 2). Viable cells produce an enhanced green £uorescence while the nonviable cells produce a red £uorescence.
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determine microbe viability in a single run is a great advantage. It is expected that applications of this sort will
expand in the future, not only in research laboratories but
also in regulatory agencies and bio-industrial areas.
References
Fig. 5. CE/LIF electropherograms generated by simultaneously monitoring the (A) green £uorescence of viable cells (520 nm) and the (B) red
£uorescence of the dead cells ( s 662 nm) obtained from Schi¡ tablets.
The uncorrected peak areas are shown in parentheses in each ¢gure.
The corrected relative peak areas show that approximately 40% of the
L. acidophilus in this sample was nonviable. See Section 2 for experimental details.
Under appropriate conditions and simultaneously monitoring the relative £uorescence intensities (Fig. 5), the ratio
of viable to nonviable cells can be obtained [11]. Analysis
shows that only about 60% of the L. acidophilus cells in
the Schi¡ tablet samples are viable (Fig. 5).
The application of high e¤ciency microbial separations
(HEMS) to commercial products has been demonstrated
for the ¢rst time. The ability to identify, quantitate, and
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