Cochu LettApplMicrobiol 2008

ORIGINAL ARTICLE
Maple sap as a rich medium to grow probiotic lactobacilli
and to produce lactic acid
A. Cochu, D. Fourmier, A. Halasz and J. Hawari
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada
Introduction
There has been an increased interest in the development
of nutraceuticals and functional foods, specifically in pro-
biotics. However, there has been an increase in the
demand for nondairy-based probiotic products. In a col-
loquium of the American Academy for Microbiology held
in Baltimore in November 2005 (report available at:
http://www.asm.org/Academy/index.asp?bid=2093) and in
a recent review (Sleator and Hill 2008), it was reported
that the use of probiotics could benefit human and ani-
mal health, specifically in the prevention or in the treat-
ment of irritable bowel syndrome, bladder and colon
cancer, urogenital infections, Clostridium difficile infec-
tions, atopic eczema, asthma and diarrhoea caused by
Rotavirus in children. Most probiotics belong to the lactic
acid bacteria (LAB) family, with many species of Lacto-
bacillus, including Lactobacillus acidophilus, Lactobacillus
casei and Lactobacillus plantarum.
In addition to the interest in lactobacilli themselves, lac-
tic acid, which is the primary metabolite of LAB, is widely
used in pharmaceutical, chemical, cosmetic, textile and
food industries. Lactic acid is also used as an acidulant, a
preservative agent, and as precursor for the production of
base chemicals or biodegradable polymers such as polylac-
tic acid (PLA; Vickroy 1985; Kharas et al. 1994). The
worldwide demand for lactic acid is increasing constantly,
and is estimated roughly to range between 130 000 and
150 000 metric tonnes per year (Mirasol 1999). Presently,
lactic acid is commercially produced by fermentation of
sugar cane (Patil et al. 2006; Timbuntam et al. 2006), corn
sugars (Mercier et al. 1992), beet molasses (Kotzamanidis
et al. 2002) and whey (Wee et al. 2006). Most of these agri-
cultural feedstocks first require a pretreatment such as
extraction or hydrolysis of their sugar content, to allow
bacterial fermentation to take place.
Forests in Eastern Canada can be considered as large
reservoirs of maple sap, which contains between 10 and
Keywords
lactic acid, lactobacilli, maple sap, probiotics,
trisaccharides.
Correspondence
Jalal Hawari, Biotechnology Research Institute,
National Research Council of Canada, 6100
Royalmount Avenue, Montreal, Que´bec, H4P
2R2, Canada.
E-mail: Jalal.Hawari@cnrc-nrc.gc.ca
2008 ⁄ 0471: received 18 March 2008,
revised 18 July 2008 and accepted: 21 July
2008
doi:10.1111/j.1472-765X.2008.02451.x
Abstract
Aims: To demonstrate the feasibility of growing lactobacilli and producing lac-
tic acid using maple sap as a sugar source and to show the importance of oli-
gosaccharides in the processes.
Methods and Results: Two maple sap samples (Cetta and Pinnacle) and puri-
fied sucrose were used as carbon sources in the preparation of three culture
media. Compared with the sucrose-based medium, both maple sap-based
media produced increased viable counts in two strains out of five by a factor
of four to seven. Maple sap-based media also enhanced lactic acid production
in three strains. Cetta sap was found to be more efficient than Pinnacle sap in
stimulating lactic acid production and, was also found to be richer in various
oligosaccharides. The amendment of the Pinnacle-based medium with trisac-
charides significantly stimulated Lactobacillus acidophilus AC-10 to grow and
produce lactic acid.
Conclusions: Maple sap, particularly if rich in oligosaccharides, represents a
good carbon source for the growth of lactobacilli and the production of lactic
acid.
Significance and Impact of the Study: This study provides a proof-of-concept,
using maple sap as a substrate for lactic acid production and for the develop-
ment of a nondairy probiotic drink.
Letters in Applied Microbiology ISSN 0266-8254
500 Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 500–507
ª 2008 The Authors

30 g l)1
of sucrose with trace amounts of glucose and
fructose. Previous studies revealed that it could be directly
used as a raw material without any pretreatment for the
production of exopolysaccharides by Enterobacter agglom-
erans (Morin et al. 1995) or for the production of poly-
hydroxybutyrate (PHB) by fermentation with Alcaligenes
latus (Yezza et al. 2007). The aim of this study was to
determine the suitability of maple sap for the production
of: (i) a high density of viable probiotic lactobacilli, and
(ii) lactic acid, a versatile chemical with potential indus-
trial and biotechnological applications.
Materials and methods
Maple sap
The maple saps were collected during the Spring of 2007,
immediately frozen and sterilized by filtration through a
0Æ22-lm filtration unit (Millipore) before use. Maple sap
designated Cetta was provided by Mr Djamel Rabia of the
‘Centre d’Expe´rimentation et de Transfert Technologique
en Ace´riculture’ (Cetta, Poheńe´gamook, Quebec, Canada).
Pinnacle maple sap was kindly provided by a personal
contact in Baldwin Mills (Quebec, Canada). Both sap
samples were harvested using tubular conduits collecting
system in the middle of March.
Chemical analysis of maple saps
All chemicals were from Sigma-Aldrich. Sucrose, glucose
and fructose were analysed using high-performance liquid
chromatography (HPLC; Waters Chromatography divi-
sion, Milford, MA, USA) equipped with an injector
model 717, a model 600 pump and a 2414 refractive
index detector. The separation was made with an ICsep
ICE-ION-300 column (Transgenomic, San Jose, CA,
USA) of 300 mm · 7Æ8 mm i.d. and an ion guard GC-
801 column (Transgenomics). The mobile phase consisted
of a solution of 0Æ035 N sulfuric acid (pH 4) flowing at
0Æ4 ml min)1
. Analyses of total organic carbon (EPA
Method 415Æ1 modified 1999), metals (EPA Method 6020,
1994) and total nitrogen (ASTM D5291, 2007) in the sap
samples were conducted by Maxxam Analytique Inc.
(Montreal, Que´bec, Canada). Oligosaccharides with vari-
ous degree of polymerization (d.p.) such as raffinose,
stachyose, maltotriose and 1-kestose were analysed by
liquid chromatography (LC)-mass spectrometry (MS)
using a Bruker micrOTOF-Q mass spectrometer attached
to a Hewlett-Packard 1200 series HPLC system (Bruker
Daltonics, Milton, Canada). The samples were injected
into a 5-lm pore size LC-NH2 column (4Æ6 mm
i.d. · 250 mm; Supelco, Bellefonte, PA, USA) at 35°C.
The solvent system was composed of a mixture of
CH3CN (70% v⁄ v)⁄ H2O (30% v⁄ v) at a flow rate of
1 ml min)1
. For mass analysis, positive electrospray ioni-
zation mode was used producing sodium adduct ions
[M+Na]+
. The mass range was scanned from 100 to
800 m⁄ z. In order to better characterize the trisaccharides
found in Cetta sap, the LC-MS method was modified by
eluting the sap through an LC-NH2 column using a sol-
vent mixture composed of acetonitrile (82%) and water
(18%) at a flow rate 1Æ5 ml min)1
. As described before,
trisaccharides were detected as their sodium adduct mass
[M+Na]+
using electrospray mass spectrometry in the
positive ionization mode.
Media
One litre of a sucrose-based medium was prepared by mix-
ing the following autoclaved solutions: 50 ml of a 400 g l)1
stock solution of veggietones pea (Oxoid), 25 ml of a
200 g l)1
stock solution of yeast extract (Difco), 10 ml of
a 200 g l)1
stock solution of K2HPO4 (Sigma), 10 ml of a
5 g l)1
stock solution of MnSO4 (Sigma), 10 ml of a
20 g l)1
stock of MgSO4 (Sigma) and 895 ml of a filtered-
sterilized sucrose solution at 22 g l)1
(purified sucrose; EM
Science, Gibbston, NJ), providing 20 g l)1
of sucrose in the
final mixture. The maple sap-based media were prepared
similarly, using 895 ml of either maple sap Cetta or Pinna-
cle instead of the 22 g l)1
sucrose solution. As determined
by HPLC, the final concentration of sucrose in the two
maple sap-based media, Cetta and Pinnacle, were 19Æ00 and
16Æ47 g l)1
, respectively. For some experiments, the Pinna-
cle-based medium was also amended with trisaccharides
such as raffinose and maltotriose, each one added at a final
concentration of 1 g l)1
.
Micro-organisms and growth conditions
Lactobacillus acidophilus R0240 and Lactobacillus helveticus
R0052 were kindly provided by Dr Thomas Tompkins
(Institut Rosell-Lallemand Inc., Montreal, Quebec, Can-
ada) and Lactobacillus rhamnosus (designated strain AC-3)
was isolated from a fresh, commercial white cheese.
Two other lactobacilli, designated as L. casei AC-8 and
L. acidophilus AC-10 were isolated from a concentrated
nondairy-based commercial probiotic product. The iden-
tity of L. rhamnosus AC-3, L. casei AC-8 and L. acidophilus
AC-10 was confirmed by comparing their 16S rDNA gene
sequence, with the 16S rDNA sequences in the NCBI data-
base (data not shown; Altschul et al. 1997).
All bacterial strains used in this study were started
from a glycerol stock and streaked on De Man Rogosa
Sharpe (MRS) agar (De Man et al. 1960). MRS plates
were incubated at 37°C under anaerobic conditions (Gas
Pack anaerobic jar system, BBL). One CFU was used to
A. Cochu et al. Growing lactobacilli in maple sap
ª 2008 The Authors
Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 500–507 501

inoculate precultures in 10 ml of MRS broth for 16 h.
Two per cent (v⁄ v) of the MRS precultures were used to
inoculate 10 ml of a maple sap (Cetta and Pinnacle) or a
sucrose-based medium, which was incubated overnight at
37°C in 15 ml conical tubes (Falcon; BD Biosciences,
Franklin Lakes, NJ) under static conditions. Subsequently,
20-ml cultures were started in either maple sap or in
sucrose-based media by adding 2% (v⁄ v) of the corre-
sponding maple sap or sucrose preculture in 20-ml serum
bottles followed by incubation for 16 h at 37°C under sta-
tic conditions. At T0 of growth, initial bacterial popula-
tion was c. 7 log CFU ml)1
for all bacteria, except for L.
acidophilus R0240, which was c. 6 log CFU ml)1
.
Analysis of culture media
Aliquots of culture medium were collected and analysed
at T0 and after 16 h of incubation as follows: (i) 0Æ1 ml
was used to determine the viable cell count (CFU), which
was carried out by diluting the samples in 0Æ1% (wt ⁄ v)
peptone water, and spread plating in duplicate on soft
MRS agar plates, which were incubated at 37°C for 48 h
under anaerobic conditions (Gas Pack anaerobic jar sys-
tem, BBL); (ii) 1 ml was used to determine A600 nm; (iii)
3 ml was centrifuged at 12 000 g for 15 min, for pH anal-
ysis and analysis of lactic acid and residual sugars by
HPLC using similar conditions as those described before.
Results
Composition of maple sap samples
Table 1 describes the composition of both Cetta and Pin-
nacle maple sap samples. As determined by HPLC, both
Cetta and Pinnacle sap contained 21Æ0–18Æ2 g l)1
of
sucrose, respectively, and low concentrations of glucose
(0Æ1–0Æ15 g l)1
) and fructose (0Æ02–0Æ08 g l)1
). Cetta and
Pinnacle saps did not markedly differ in their total
organic carbon and total nitrogen concentrations. How-
ever, the two sap samples differed in their nitrate, sulfate
and sodium content. For example, the concentration of
nitrate and sulfate were 5- and 20-fold higher in Cetta
sap, but in Pinnacle the concentration of sodium was
50-fold lower (data not shown). Calcium, magnesium and
manganese were also present in higher amounts in Cetta
sap than in Pinnacle sap; however, the potassium contents
were slightly similar in both saps (data not shown).
Characterization of oligosaccharides in maple sap
LC-MS analysis of Cetta and Pinnacle saps revealed the
presence of oligosaccharides with d.p. ranging from 3 to
5 (Fig. 1). The area of the peaks corresponding to oligo-
saccharides with a d.p. of 4 and 5 were found to be
greater in Cetta than in Pinnacle sap, but those corre-
sponding to trisaccharides (d.p. 3) were found to be c. 11
times greater in Cetta than in Pinnacle sap (5Æ6 · 107
and
4Æ8 · 106
, respectively) (Fig. 1).
To better characterize the d.p. 3 oligosaccharides shown
in Fig. 1, a modified LC-MS method was employed to
improve separation of overlapping signals to enhance
their detection. Figure 2a represents a typical LC-MS
chromatogram of trisaccharides in Cetta maple sap. The
peak at 25Æ2 min exhibits a sodium adduct mass
[M+Na]+
at 527 Da with a retention time (r.t.) similar to
that of 1-kestose (b-d-Fruf-(2 fi 1)b-d-Fruf(2 fi 1)a-
d-Glup). The minor LC-MS peak was observed with a
r.t. at 30Æ1 min and a [M+Na]+
at 527 Da and matched
the two trisaccharides, raffinose (O-a-galactopyrano-
syl(1 fi 6)a-d-glucopyranosyl-b-d-fructofuranoside) and
maltotriose (O-a-dd-glucopyranosyl-(1 fi 4)-O-a-d-
glucopyranosyl-(1 fi 4)-d-glucose), with each showing
the same r.t. at 30Æ1 min and [M+Na]+
at 527 Da. The
identities of these sugars and the major compound
eluting at 21Æ5 min will be discussed latter.
Growing lactobacilli in sucrose- and maple sap-based
media
The growth of L. acidophilus R0240, L. helveticus R0052
and L. acidophilus AC-10 resulted in a drop in pH (from
7Æ2 to below 4Æ5, not shown), and significant sucrose con-
sumption (1Æ3–6Æ0 g l)1
; Table 2). Whichever medium
was used, with the exception of L. acidophilus R0240 that
had low final bacterial counts, the viable counts of all
other strains reached c. 9 log CFU ml)1
after 16 h of fer-
mentation at 37°C (Fig. 3a). Extending the fermentation
time from 16 to 24 h did not result in an increased viable
count (data not shown). While L. rhamnosus AC-3,
L. acidophilus R0240 and L. casei AC-8 grew similarly in
the sucrose- and maple-based culture media, L. helveticus
R0052 and L. acidophilus AC-10 grew four to seven times
more in the maple sap-based media (Fig. 3a) and also
produced more lactic acid (1Æ89–4Æ94 g l)1
) than the
other strains (Fig. 3b). No other volatile organic acids
Table 1 Composition (in g l)1
) of the maple sap samples used
Sap component
Cetta maple sap
(Pohenegamook,
Quebec, Canada)
Pinnacle maple sap
(Balwin Mills,
Quebec, Canada)
Fructose 0Æ080 0Æ026
Glucose 0Æ145 0Æ098
Sucrose 21Æ001 18Æ201
Total organic carbon 10Æ300 9Æ210
Total nitrogen <0Æ100 <0Æ100
Growing lactobacilli in maple sap A. Cochu et al.
ª 2008 The Authors

such as acetic acid were detected in the cultures. For all
cultures, except for L. casei AC-8, Cetta maple sap led to
higher lactic acid production than the Pinnacle maple
sap. It is interesting to note that L. helveticus R0052 and
L. acidophilus AC-10 both consumed increased amounts
of sucrose when grown in maple sap-based media
(Table 2). On the other hand, Table 2 shows that the
three other strains (AC-3, R0240 and AC-8) generally
consumed comparable amounts of sucrose in sucrose-
based and in Pinnacle-based media, but much less in
Cetta-based medium. To understand what factors in
maple sap could contribute to increased growth, sucrose
consumption and lactic acid production, complementary
experiments of bacterial growth in maple sap-based media
were performed.
Indeed, the two sap samples, Cetta and Pinnacle, varied
in their chemical compositions (Table 1) and we attrib-
uted the variation in lactic acid production (no significant
difference was observed on bacterial growth) between the
two samples as due to cumulative effect of maple sap
components. To demonstrate the effect of oligosaccha-
rides in promoting lactobacilli growth and in increasing
their lactic acid production, we monitored the disappear-
ance of the oligosaccharides initially present in the Cetta
maple-based medium. After 16 h of incubation, LC-MS
analysis showed that L. helveticus R0052 and L. acidophi-
lus AC-10 consumed c. 70% of the d.p. 4 oligosaccharide
appearing at 19Æ2 min and 50% of the major d.p. 3 oligo-
saccharides shown in Fig. 2a (data not shown). Since Pin-
nacle sap contained less oligosaccharides, we thus
amended the Pinnacle sap-based medium with a mixture
of two d.p. 3 oligosaccharides, raffinose (1 g l)1
) and
maltotriose (1 g l)1
) and used it to grow L. acidophilus
AC-10. Because A600 nm was previously found to perfectly
correlate with CFU (data not shown), this parameter was
used to measure bacterial growth. The lactobacilli grew
approximately three times more in the Pinnacle medium
amended with raffinose and maltotriose, and produced
double the concentration of lactic acid than when grown
in the nonamended Pinnacle medium (Table 3). It should
be noted that the average lactic acid concentration
obtained in the nonamended Pinnacle sap-based medium
presented in Table 3 is different from the average lactic
acid concentration previously shown Fig. 3b (2Æ95 and
1Æ89 g l)1
, respectively). Despite the fact that the 20-ml,
16-h culture was done similarly, the incubation times of
the precultures varied between the two experiments
explaining the variation observed.
d.p.3
d.p.3
d.p.4
d.p.4
d.p.5
d.p.3
d.p.3
d.p.4
d.p.5
0 10 20 30 40 50 Time (min)
0·00
0·25
0·50
0·75
1·00
1·25
x104
Intens·
(a)
(b)
0 10 20 30 40 50 Time (min)
0·00
0·25
0·50
0·75
1·00
1·25
x104
Intens·
Figure 1 Liquid-chromatography–mass
spectrometry profile of the oligosaccharides
with degree of polymerization (d.p.) from
3 to 5 in Cetta (a) and Pinnacle (b) maple
saps obtained by ESI-Qq-TOF mass
spectrometer using positive ionization mode.
ª 2008 The Authors

Discussion
In this study, we have investigated the use of maple sap
as a new feedstock for the production of probiotic lacto-
bacilli and lactic acid. The LAB strains were selected
based on their ability to yield high concentrations of via-
ble cells when grown on maple sap-based media (Fig. 3a).
Our results clearly demonstrate that (i) maple sap consti-
tutes a good carbon source; (ii) supported the growth of
L. rhamnosus AC-3, L. helveticus R0052, L. casei AC-8 and
L. acidophilus AC-10 at c. 9 log CFU ml)1
and (iii)
allowed L. helveticus R0052 and L. acidophilus AC-10 (the
best lactic acid producers of the study) to produce up to
5 g l)1
of lactic acid after 16 h of fermentation at 37°C in
Cetta sap-based medium (Fig. 3b). Figure 3 also shows
that L. rhamnosus AC-3 and L. casei AC-8 are good
candidates for production of probiotics because lactic
acid if produced would be in trace amounts. Viable bacte-
rial counts and lactic acid production were correlated
with signiﬁcant sucrose consumption. These results show
that maple sap was favourable compared with other
sucrose-based feedstocks, such as beet juice and soymilk
0 10 20 30 40 50 Time (min)
0·0
0·5
1·0
1·5
x105
0 10 20 30 40 50 Time (min)
0·0
0·5
1·0
1·5
x105
0·0
0·5
1·0
1·5
x105
1-Kestose spiked maple sap
Raffinose spiked maple sap
Maltotriose spiked maple sap
0 10 20 30 40 50 Time (min)
0 10 20 30 40 50 Time (min)
0·0
0·5
1·0
1·5
(a)
(b)
(c)
(d)
x105
Figure 2 Extracted ion chromatograms
([M+Na]+
at m ⁄ z 527) of the trisaccharides
from Cetta maple sap (a), 1-kestose spiked
(b), rafﬁnose spiked (c) and maltotriose spiked
(d) Cetta maple sap.
ª 2008 The Authors

containing 58 and 28 g l)1
of sucrose respectively, each of
which were shown to support the growth of lactobacilli
(8–9 log CFU ml)1
) and the production of lactic acid
(2Æ3–5Æ3 g l)1
) under similar culture conditions, i.e. batch
cultures conducted without pH control or lactic acid
removal (Garro et al. 1998; Yoon et al. 2005).
The increased lactic acid production and sucrose con-
sumption by L. helveticus R0052 and L. acidophilus AC-10
in Cetta maple sap compared with Pinnacle maple sap led
us to search for variations in their chemical compositions.
The maple sap samples came from two different regions
of Quebec and literature indicates that the location and
Table 2 Sucrose consumption (in g l)1
) by various lactobacilli after 16 h of fermentation in puriﬁed sucrose-based medium or in maple sap-based
media*
Strains
Sucrose-based
medium
Cetta-based
medium
Pinnacle-based
medium
Lactobacillus rhamnosus AC-3 1Æ05 ± 0Æ13 0Æ00 ± 0Æ04 1Æ01 ± 0Æ16
Lactobacillus acidophilus R0240 2Æ41 ± 0Æ29 1Æ26 ± 0Æ09 2Æ47 ± 0Æ13
Lactobacillus helveticus R0052 3Æ42 ± 0Æ04 5Æ64 ± 0Æ11 4Æ41 ± 0Æ08
Lactobacillus casei AC-8 0Æ92 ± 0Æ11 0Æ48 ± 0Æ09 0Æ95 ± 0Æ24
Lactobacillus acidophilus AC-10 3Æ20 ± 0Æ10 5Æ95 ± 0Æ03 4Æ57 ± 0Æ12
*The initial concentrations of sucrose in sucrose-, Cetta- and Pinnacle-based media were respectively, 20Æ0, 19Æ0 and 16Æ5 g l)1
. Glucose and fruc-
tose were totally consumed by all cultures.
7
8
9
10(a)
(b)
L. rhamnosus AC-
3
L. acidophilus
R0240
L. helveticus
R0052
L. casei AC-8 L. acidophilus AC-
10
logCFUml–1
0·00
0·83
0·92 0·75
0·35
1·98
4·94 4·82
0·00
1·20
1·98 1·89
0·00 0·00 0·00
0
2
4
6
L· rhamnosus AC-
3
L· acidophilus
R0240
L· helveticus
R0052
L· casei AC-8 L· acidophilus AC-
10
Lacticacidproduced(gl–1
)
Figure 3 Bacterial viable count (a) and lactic acid produced (b) by lactobacilli in sucrose-based (black), Cetta (white) or Pinnacle (grey) sap-based
media after 16 h of fermentation at 37°C. CFU, colony-forming units; L., Lactobacillus. Values indicate averages from two distinct cultures and
bars represent the SE.
ª 2008 The Authors

several other factors that include, the age of maple trees,
period and method of maple harvest, will largely influ-
ence sap composition in terms of sugars, minerals, phe-
nolic compounds, vitamins and organic acids (Morselli
1975; Kuentz et al. 1976; Stuckel and Low 1996). Despite
the fact that some differences between the two saps were
noted in the concentrations of nitrate, sulfate, sodium,
magnesium and manganese, the amendment of sap with
culture medium components such as phosphate, yeast
extract, veggietones pea, manganese and magnesium likely
rendered these differences insignificant. Also because
sucrose was always partially consumed by the lactobacilli
cultures, the difference in sucrose concentration does not
explain the difference in growth and lactic acid produc-
tion observed in the two sap samples, Cetta and Pinnacle.
However, the most significant difference observed
between the two maple sap samples was in their content
of oligosaccharides. Both maple saps revealed the presence
of oligosaccharides with a d.p. ranging from 3 to 5. We
found that Cetta sap that contained a higher content of
oligosaccharides, particularly trisaccharides, showed a
slightly higher bacterial growth and higher yield of lactic
acid than Pinnacle. For example, Cetta sap was found to
contain 1-kestose as one of the two major trisaccharides
as confirmed by comparison with a reference standard
(Fig. 2a,b). The second major trisaccharide was tentatively
identified as neokestose (Fig. 2a) based on previous
assignment made by Haq and Adams (1961) who also
reported neokestose as a major trisaccharide in maple
sap. Whereas the minor LC-MS peak with a r.t. at
30Æ1 min was tentatively identified as raffinose as con-
firmed by Porter et al. (1954) who reported the presence
of this sugar based on detailed structural analysis of the
trisaccharide in maple sap. However, more recently Bazi-
net et al. (2007) suggested the trisaccharide to be a malto-
triose based on HPLC ⁄refractive index analysis alone.
Further confirmation to our assignments of the three tri-
saccharides was obtained by proper spiking of each ana-
lyte separately during HPLC analysis (Fig. 2b–d).
Literature reports indicated that raffinose-like oligosac-
charides could enhance the acidification rate and the pop-
ulation levels of strains of L. acidophilus and
Bifidobacterium lactis (Martıńez-Villaluenga et al. 2005).
More recently, it was shown that also fructo-oligosaccha-
rides can enhance the production of various bacteriocins
by LAB (Chen et al. 2007) and the soybean fructo-oligo-
saccharides, inulin and raffinose, are able to enhance the
growth of various probiotic bacteria (Su et al. 2007). In
the present study, when the Pinnacle-based medium was
amended with the two trisaccharides, raffinose (1Æ0 g l)1
)
and maltotriose (1Æ0 g l)1
), L. acidophilus AC-10 grew
approximately three times faster and the lactic acid yield
increased from 2Æ95 g l)1
to 5Æ95 g l)1
.
In conclusion, our results revealed that maple sap can
be considered as a remarkable renewable feedstock for
developing a nondairy drink with probiotic lactobacilli.
Among the strains tested, L. rhamnosus AC-3 and L. casei
AC-8 represented the best choices for the probiotic drink
owing to their high viable cells count and low lactic acid
production. Finally, maple sap-based media may serve as
a convenient substrate for significant lactic acid produc-
tion by L. helveticus R0052 and L. acidophilus AC-10
without pretreatments of maple sap.
Acknowledgements
The authors wish to thank Ste´phane Deschamps, Chan-
tale Beaulieu, Louise Paquet, Karine Trudel and Alain
Corriveau for their excellent technical assistance. They
also thank Punita Mehta for the revision of the
manuscript.
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Amended Pinnacle sap 1Æ60 ± 0Æ066 5Æ95 ± 0Æ260
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ª 2008 The Authors

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