Production of beta-glucosidase and hydrolysis of isoflavone phytoestrogens by Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus casei in soymilk.

Abstract

The study determined beta-glucosidase activity of commercial probiotic organisms for hydrolysis of isoflavone to aglycones in fermenting soymilk. Soymilk made with soy protein isolate (SPI) was fermented with Lactobacillus acidophilus LAFTI L10, Bifidobacterium lactis LAFTI B94, and Lactobacillus casei LAFTI L26 at 37 degrees C for 48 h and the fermented soymilk was stored for 28 d at 4 degrees C. beta-Glucosidase activity of organisms was determined using rho-nitrophenyl beta-D-glucopyranoside as a substrate and the hydrolysis of isoflavone glycosides to aglycones by these organisms was carried out. The highest level of growth occurred at 12 h for L. casei L26, 24 h for B. lactis B94, and 36 h for L. acidophilus L10 during fermentation in soymilk. Survival after storage at 4 degrees C for 28 d was 20%, 15%, and 11% greater (P < 0.05) than initial cell counts, respectively. All the bacteria produced beta-glucosidase, which hydrolyzed isoflavone beta-glycosides to isoflavone aglycones. The decrease in the concentration of beta-glycosides and the increase in the concentration of aglycones were significant (P < 0.05) in the fermented soymilk. Increased isoflavone aglycone content in fermented soymilk is likely to improve the biological functionality of soymilk.

Full text

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M:FoodMicrobiology
&Safety
JFS M: Food Microbiology and Safety
Productionofβ-GlucosidaseandHydrolysis
ofIsoflavonePhytoestrogensbyLactobacillus
acidophilus,Bifidobacteriumlactis,
andLactobacilluscasei inSoymilk
O.N. DONKOR AND N.P. SHAH
ABSTRACT: The study determined β-glucosidase activity of commercial probiotic organisms for hydrolysis of
isoflavonetoaglyconesinfermentingsoymilk.Soymilkmadewithsoyproteinisolate(SPI)wasfermentedwithLac-
tobacillus acidophilus LAFTI?L10, Bifidobacterium lactis LAFTI?B94, and Lactobacillus casei LAFTI?L26 at 37◦C
for 48 h and the fermented soymilk was stored for 28 d at 4◦C. β-Glucosidase activity of organisms was determined
using ρ-nitrophenyl β-D-glucopyranoside as a substrate and the hydrolysis of isoflavone glycosides to aglycones by
these organisms was carried out. The highest level of growth occurred at 12 h for L. casei L26, 24 h for B. lactis B94,
and 36 h for L. acidophilus L10 during fermentation in soymilk. Survival after storage at 4◦C for 28 d was 20%, 15%,
and 11% greater (P < 0.05) than initial cell counts, respectively. All the bacteria produced β-glucosidase, which hy-
drolyzed isoflavone β-glycosides to isoflavone aglycones. The decrease in the concentration of β-glycosides and the
increaseintheconcentrationofaglyconesweresignificant(P <0.05)inthefermentedsoymilk.Increasedisoflavone
aglycone content in fermented soymilk is likely to improve the biological functionality of soymilk.
Keywords: aglycone, β-glucosidase, isoflavones, soy protein isolate, viability
Introduction
I
tages over dairy, milk including reduced level of cholesterol and
saturated fat as well as the absence of lactose. Furthermore, soy
products contain the isoflavone phytoestrogens with potential an-
ticarcinogenic activity (Uzzan and Labuza 2004). Their antioxidant
ability may also prevent oxidative damage in living tissue (Wei and
others 1995; Ruiz-Larrea and others 1997). Isoflavones occur nat-
urally in plants, in particular soybeans. Isoflavones are a part of
the diphenol compounds, which are structurally and functionally
similar to the human estrogen, estradiol, but are much less potent
than estradiol (Tsangalis and others 2002). Because of this similar-
ity, isoflavones were suggested to have preventive effects for many
kinds of hormone-dependent diseases (Uzzan and Labuza 2004).
Isoflavones in soy proteins in most soy foods are conjugated with
sugars.Theβ-glucosideformsarenotabsorbedandrequirehydrol-
ysis for bioavailability and subsequent metabolism. Hydrolysis oc-
curs along the entire length of the intestinal tract by the action of
boththebrushbordermembrane-andthebacterialβ-glucosidases
(Setchell and others 2002). The aglycones are released and further
metabolism of daidzein and genistein takes place. Intestinal bio-
transformations include dehydroxylation, reduction, C-ring cleav-
age, and demethylation. These reactions take place distally and
presumably in the colon. However, the types of intestinal bacteria
involved in isoflavone conversion to bioactive forms and the effec-
tiveness of this microbial biotransformation are not well known.
n addition to serving as a delivery medium for probiotic organ-
isms to the consumer, soymilk has several nutritional advan-
MS 20070569 Submitted 7/23/2007, Accepted 8/22/2007. Authors are with
School of Molecular Sciences, Victoria Univ., Werribee Campus, P .O. Box
14428, Melbourne, Vic 8001, Australia. Direct inquiries to author Shah
(E-mail: Nagendra.Shah@vu.edu.au).
Bifidobacterium and Lactobacillus are the predominant members
of the intestinal microflora; however, Bifidobacterium strains are
widely studied for the production of β-glucosidase (Tochikura and
others 1986; Tsangalis and others 2002; Hsu and others 2005) lead-
ing to transformation of isoflavones to bioavailable and bioac-
tive forms (Tsangalis and others 2002, 2004; Wei and others 2007).
However, production of β-glucosidase by other groups of bacte-
ria has not been studied. Therefore, it will be interesting to study
other groups of bacteria for the production of β-glucosidase us-
ing commercial probiotic organisms (L. acidophilus L10, B. lac-
tis B94, and L. casei L26) and biotransformation of isoflavone
aglycones by these microorganisms in soymilk. Probiotics are de-
fined as “live microorganisms which when administered in ade-
quate amounts confer a health benefit on the host” (FAO/WHO
2002).
The aims of this study were to evaluate β-glucosidase activity of
selected probiotic organisms (L. acidophilus L10, B. lactis B94, and
L. casei L26) for hydrolysis of isoflavone to aglycones in soymilk fer-
mented at 37◦C for 48 h, and to assess cell population and organic
acid production during fermentation of soymilk at 37◦C for 48 h
and storage at 4◦C for 28 d.
Materials and Methods
Propagation of cultures
Lactobacillus acidophilus LAFTI?L10, Bifidobacterium lactis
LAFTI?B94, and L. casei LAFTI?L26 were provided by DSM Food
Specialties (Moorebank, NSW, Australia). These microorganisms
have been reported to have probiotic properties (Crittenden and
others 2005). Each culture was stored at −80◦C. Sterile 10-mL
aliquots of MRS were inoculated with 1% of each culture and in-
cubated at 37◦C for 20 h. For propagation of Bifidobacterium,
C ?2007 Institute of Food Technologists
doi: 10.1111/j.1750-3841.2007.00547.x
Further reproduction without permission is prohibited
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Production of ?-glucosidase and hydrolysis.. .
sterile MRS broth was supplemented with 0.05% L-cystein HCl to
provide anaerobic conditions and stimulate growth (Ravula and
Shah 1998). After 3 successive transfers in MRS broth, the ac-
tivated organisms were transferred at 5% v/v to 10-mL aliquots
of sterile soymilk supplemented with 2% glucose and 1% yeast
extract. After a 2nd transfer in sterile soymilk without yeast ex-
tract the cultures were ready for the production of fermented
soymilk.
Soymilk manufacture
Soymilk was prepared as per the method of Tsangalis and oth-
ers (2004) by mixing 4% soy protein isolate (SPI) SUPRO?590 IP
(proximate composition: moisture 6.0%, protein, dry basis 90.0%,
fat 1.5%, ash 5.0%, carbohydrate ≤ 1.0%), supplied by SOLAE Co.
(Chatswood,NSW,Australia)(w/v)indeionizedwater.Forreconsti-
tution, deionized water was heated to 40◦C prior to addition of SPI
powder, followedbyheatingthemixtureto50◦Cwith constant stir-
ring for 30 min to dissolve solid particles. A 11.4 L batch of soymilk
waspreparedanddispensedinto19glassbottlescontaining300mL
each. The bottles containing soymilk were sterilized by autoclaving
at 121◦C for 15 min.
Fermentation of soymilk with microorganisms
Twosetsof6glassbottleseachcontaining300mLsterilesoymilk
were aseptically inoculated with L. acidophilus L10 at 5% (v/v) and
incubated at 37◦C for 48 h. The bottles were labeled 0, 6, 12, 24,
36, and 48 h in order to facilitate withdrawing of aliquots at 0,
6, 12, 24, 36, and 48 h of fermentation. Aliquots of 50 mL from
each of the 6 bottles were taken at 0, 6, 12, 24, 36, and 48 h of in-
cubation. Each aliquot was divided into 20- and 30-mL portions
in sterile 50-mL screw top falcon tubes for determination of β-
glucosidase activity and pH and for enumeration of cell counts,
whereasthe30-mLportionswereimmediatelystoredat−80◦Cand
later freeze dried using a Dynavac?FD 300 freeze drier (Rowville,
Vic., Australia) for the analysis of isoflavones. The 2nd set of 6 bot-
tles of fermented soymilk was stored at 4◦C for 28 d for deter-
mination of β-glucosidase activity and pH and for enumeration
of bacteria. The experiments were repeated for B. lactis B94 and
L. casei L26.
β-Glucosidase activity in soymilk
β-Glucosidase activity of the 3 microorganisms was determined
in soymilk during fermentation at 37oC for 6, 12, 24, 36, and
48 h. Five milliliter aliquot samples were taken at 0, 6, 12, 24, 36,
and 48 h and the enzyme activity was determined immediately.
The β-glucosidase activity was determined by measuring the rate
ofhydrolysisofρ-nitrophenylβ-D-glucopyranoside(ρNPG)(Sigma
Chemical Co., St. Louis, Mo., U.S.A.) as per the methods described
previously (Scalabrini and others 1998; Otieno and others 2006)
with some modifications. Five hundred microliters of 5 mM ρNPG
prepared in 100 mM sodium phosphate buffer (pH 7.0) was added
to 5 mL of each aliquot sample and incubated at 37◦C for 30 min.
The reaction was stopped by the addition of 250 μL cold 0.2 M
sodium carbonate (4◦C). The resulting mixture was centrifuged
at 14000 × g for 30 min using an Eppendorf centrifuge (model
5415C;CrownScientific,Melbourne,Australia)andfilteredthrough
a 0.45-μm membrane filter (Schleicher & Schuell GmbH, Dassel,
Germany). The amount of ρ-nitrophenol released was measured
using a spectrophotometer (Pharmacia LKB?Novospec II, Upp-
sala, Sweden) at 420 nm. One unit of enzyme activity is defined
as the amount of β-glucosidase activity that released 1 μmol of
ρ-nitrophenol from the substrate ρNPG per milliliter per minute
under the assay conditions.
Enumeration of bacterial population
Cell populations of L. acidophilus L10, B. lactis B94, and L. casei
L26 were determined as described previously (Donkor and others
2005). Briefly, 1 g of each fermented soymilk sample was added to
9 mL of sterile 0.15% (w/v) bacteriological peptone (Oxoid) and
water diluent and vortexed (model MT 19, Chiltern Scientific,
Salmond Smith Biolab Ltd., Auckland, New Zealand) for 30 s. The
resulting suspension was serially diluted in sterile 0.15% (w/v) pep-
tone water (Oxoid) and 1 mL of the appropriate dilution was used
for selective enumeration by the pour plate technique. The cell
growth of each organism was assessed by enumerating bacterial
population after 6, 12, 24, 36, and 48 h of fermentation of soymilk
on MRS agar (Amyl media). Anaerobic jars and gas generating kits
(Anaerobic system BR 38, Oxoid Ltd., Hampshire, England) were
used for creating anaerobic conditions. Duplicate plates were in-
cubated anaerobically for 72 h at 37◦C for L. acidophilus, L. casei,
and Bifidobacterium spp. Plates containing 25 to 250 colonies were
counted and recorded as colony forming units (CFU) per gram of
the fermented soymilk.
pH measurements
Changes in pH were monitored during fermentation of soymilk
at 0, 6, 12, 24, 36, and 48 h using a pH meter (HANNA Instruments,
Singapore).
Production of organic acids in fermenting soymilk
During culture growth, the main metabolic products are organic
acids, particularly lactic and acetic acids. The concentrations of
these acids were measured according to the method of Donkor
and others (2005) using high-performance liquid chromatrogra-
phy (HPLC). The HPLC (Varian Associates, Walnut Creek, Calif.,
U.S.A.) comprised a solvent delivery system (model 9100) con-
necting with an auto-sampler (model 9012), a UV light detector
(model 9050) and an organic acid analysis column (Aminex HPX-
87H,300×7.8mm,Bio-RadLab,Richmond,Calif.,U.S.A.).Themo-
bile phase was 0.001 M H2SO4with a flow rate set at 0.6 mL/min
and the temperature of the column was set at 65◦C. Organic
acids were detected at 220 nm. For determination of organic acids,
2.5 g samples were mixed with 50 μL of 15.8 N HNO3and 1.0 mL
of 0.001 M H2S04before subjecting a 1.5-mL aliquot of the mix-
ture to centrifugation at 14000 × g for 30 min at room temperature
(∼20◦C). The supernatant was filtered through a 0.45-μm mem-
brane filter (Schleicher & Schvell, Dassel, Germany) and 20 μL of
the filtrate was injected into the HPLC system.
Isoflavone extraction
The extraction of isoflavones from fermented and non-
fermented soymilk was performed in triplicate using the method
described by Otieno and others (2006) with some modifications.
Briefly, 50-mL aliquots of methanol were added to 1.0 g freeze dried
sample in a 150-mL round bottom flask and refluxed on a heating
mantle for 1 h. The mixture was poured into a 50-mL centrifuge
tube and was centrifuged at 4000 × g for 30 min at 10◦C using
a Sorvall RT7 refrigerated centrifuge (Newtown, Conn., U.S.A.). A
2-mL aliquot of the supernatant was withdrawn and mixed with
50 μl of internal standard (ISTD) flavone solution (0.2 mg/mL) and
evaporated to dryness using the speed vac concentrator connected
to Savant refrigerated trap and vacuum pump (model SC110, Sa-
vant Instruments Inc., Farmingdale, N.Y., U.S.A.). The dried sample
was dissolved in 1 mL mobile phase (0.05% TFA in 50% of 100 mM
ammonium acetate and 50% methanol) and the resulting solu-
tion was centrifuged at 14000 × g for 30 min using an Eppendorf
centrifuge (model 5415C; Crown Scientific). The supernatant was
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Production of ?-glucosidase and hydrolysis. . .
filtered through a 0.45-μm membrane filter (Schleicher & Schuell
GmbH) into an HPLC vial.
Isoflavone standards
Isoflavone standard stock solutions were prepared by dissolv-
ing genistein, daidzein, genistin, and flavone (Sigma) in HPLC
grade methanol and glycitein, daidzin, and glycitin were dissolved
in ethanol (Sigma) to give a 1 mg/mL concentration. Calibration
curves were prepared for each standard with 6 different concentra-
tions (0, 20, 40, 60, 80, and 100 μg/mL). A mixture of the standards
was also analyzed and used for identification purposes.
HPLC with electrochemical detection
of isoflavone in fermented soymilk
For electrochemical determination of isoflavones in soymilk,
a highly sensitive HPLC-ECD method was used as described
previously (Klejdus and others 2004) with some modifications.
Isoflavone extracted from unfermented (control) and fermented
soymilk samples were profiled on a reversed-phase Varian HPLC
(Varian Analytical Instruments) system. The HPLC system for elec-
trochemical detection consisted of the following components: a
Varian model 9100 auto sampler, a Varian model 9012 solvent de-
livery system, injector, a Metrohm model 656 amperometric, and
641 VA detector with a 1 μL cell with glassy carbon working and
auxilliary electrode, and a Ag/AgCl reference electrode (Metrohm,
Herisau,Switzerland).Sampleswereappliedusinga20μLinjection
loop and separation of isoflavone compounds was achieved using
an Alltima HP C18HL, 5 μm, 250 x 4.6 mm column and a guard col-
umn(AlltechAssociates,Inc.,Deerfield,Ill.,U.S.A.).Theisoflavones
were eluted by isocratic runs of 40 min with mobile phase (0.05%
TFA in 50% of 100 mM ammonium acetate and 50% methanol) at a
Figure 1---Changes in bacteria
population (? CFU/g) and
concentrations of β-glycosides and
aglycones (μmol per mL) in soymilk
undergoing fermentation by L.
acidophilus L10, B. lactis B94, and
L. casei L26 for 6, 12, 24, 36, and
48 h at 37◦C. L10 = L. acidophilus,
B94 = B. lactis, L26 = L. casei (n =
6, Error bars represent means ±
standard error).
flow rate of 1 mL/min and room temperature (∼20◦C), a sensitiv-
ity range of 0.1 to 50 nA, and an electrochemical detection at +800
mV. All solvents were filtered through a 0.45-μm membrane filter
(Schleicher & Schuell GmbH) and degassed by ultrasound and he-
lium sparging.
Retention times of standards of isoflavones eluted as single
peaks (glycitin, daizin, genistin, daizein, glycetein, and genistein)
ranged between 2.57 to 17.48 min, and the peak areas were used
for quantitation. Calibration runs were prepared for each analyti-
cal series with external standards in the range of 20 to 200 ng/mL
of isoflavones. The calibration curves were linear with a regression
coefficient of 0.998.
Statistical analysis
All results obtained were analyzed as a split plot in time design
using the general linear model (GLM) procedure of the SAS sys-
tem (SAS 1996). The univariate ANOVA test was validated by ful-
filling the Huynh-Feldt (H-F) condition (Littell and others 1998).
Where appropriate, 1-way ANOVA and correlational analysis were
employed using Microsoft?Excel StatProTM(Albright and oth-
ers 1999) and the multicomparison of means was assessed by
Tukey’s test. The statistical level of significance was preset at 0.05.
All experiments were replicated and subsampled at least twice
(n = 6).
Results
Cell growth during fermentation and storage
Cell populations of L. acidophilus L10, B. lactis B94, and L.
casei L26 in soymilk during fermentation at 6, 12, 24, 36, and
48 h at 37◦C are shown in Figure 1. The highest viable counts
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Production of ?-glucosidase and hydrolysis.. .
during fermentation occurred at 12 h for L. casei L26, 24 h for
B. lactis B94, and 36 h for L. acidophilus L10. Subsequently the
populations declined (P > 0.05) slowly for each microorganism
after growth (Figure 1). Soymilk fermented with L. acidophilus
L10, B. lactis B94, and L. casei L26 and stored at 4◦C for 28 d
showed an increased trend of bacterial growth, with L. acidophilus
L10 and B. lactis B94 exhibiting increased population by 20% and
14%, respectively (Figure 2). Even though the different fermenta-
tion times influenced bacterial growth during storage 4◦C, there
was a general decline in cell populations during storage from 21 to
28 d.
Decline in pH during fermentation and storage
There was a decline in pH during fermentation at 6, 12, 24, 36,
and 48 h at 37◦C (data not shown). Each batch of soymilk showed
consistent decrease in pH from the initial pH of 6.58 during 48 h in-
cubation. B. lactis B94, and L. casei L26 presented a lower but simi-
lardropinpH(0.73pHunits)comparedtoL.acidophilusL10which
showed a decline of 0.59 pH units at the end of storage. The trend
was similar and was not significantly different for samples from all
Figure 2---Percentage
cell counts (% CFU per
g) in soymilk which
had undergone
fermentation by L.
acidophilus L10, B.
lactis B94, and L.
casei L26 for 24 and
48 h at 37◦C and
stored at 4◦C for 28 d.
L10 = L. acidophilus,
B94 = B. lactis, L26 =
L. casei (Error bars
represent a pooled
standard error of the
mean; n = 6).
Figure 3---Lactic and acetic acid concentrations produced in soymilk undergoing fermentation by L. acidophilus L10,
B. lactis B94, and L. casei L26 for 6, 12, 24, 36, and 48 h at 37◦C as single cultures. L10 = L. acidophilus, B94 = B.
lactis, L26 = L. casei (error bars represent a pooled standard error of the mean; n = 6; SEM for lactic and acetic acid
= 0.03 and 0.01 mg/mL, respectively).
5 fermentation times during the 28 d of storage period (data not
shown).
Organic acids concentration during
fermentation and storage
The concentration of lactic and acetic acids in fermented
soymilk produced by L. acidophilus L10, B. lactis B94, and L. ca-
sei L26 is shown in Figure 3. In general, the lactic acid concentra-
tion was higher (P < 0.05) than acetic acid for all treatments and
times. The concentration of lactic acid in soymilk increased consis-
tently during 48 h of fermentation for all the organisms, with L. aci-
dophilus L10 producing the lowest concentration of lactic acid but
not significantly (P < 0.05) different from B. lactis B94. However,
in comparison to L. acidophilus L10, the production of lactic acid
by L. casei L26 was significantly (P < 0.05) higher at the end of fer-
mentation. Even though the organic acid content increased slightly
during storage at 4◦C for 28 d, there was no significant difference
between cultures for lactic and acetic acids (data not shown). Since
thegrowthwasslowduringstorage,theproductionoforganicacids
was lower (P > 0.05) which resulted in a minimal decline in pH.
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Production of ?-glucosidase and hydrolysis. . .
Consequently fermented soymilk did not set during 28 d storage at
4◦C.
β-glucosidase activity
The β-glucosidase activity of L. acidophilus L10, B. lactis B94,
and L. casei L26 in soymilk before and after fermentation at 37◦C
is shown in Figure 4. The maximum yield of β-glucosidase activ-
ity varied (P < 0.05) between all 3 organisms. L. acidophilus L10
showed increased enzyme activity at 36 h of fermentation whereas
B. lactis B94 and L. casei L26 attained maximum enzyme activities
at 24 and 12 h, respectively. During storage at 4◦C, all microorgan-
isms showed maximum β-glucosidase activity ranging from 0.117
to0.204μmol/mLatd21irrespectiveoffermentationtime.Overall,
B. lactis B94 exhibited the highest β-glucosidase activity followed
by L. acidophilus L10 during storage (data not shown). The corre-
lation between β-glucosidase activity and growth of microorgan-
ismsrangedfrom0.70forL.acidophilusL10to0.79forB.lactisB94,
which further suggested the effects of growth of microorganisms in
soymilk on β-glucosidase activity.
Hydrolysis of isoflavone compounds in
fermented soymilk by microorganisms
The changes in isoflavone concentration in relation to increas-
ing bacteria population in soymilk during 48 h fermentation at
37◦C with L. acidophilus L10, B. lactis B94, and L. casei L26, re-
spectively, is shown in Figure 1. The maximum concentration of
aglyconesproducedcorrespondedtothemaximumcellpopulation
of each microorganism. In general, the concentration of isoflavone
aglycones increased (P < 0.05) and at the same time the concentra-
tionofβ-glycosideswasreduced(P <0.05)comparedtothatinun-
fermented soymilk. However, the concentrations of malonyl- and
acetyl-glycosides were relatively low and there was no significant
change (P > 0.005) during the 48 h fermentation (data not shown).
Therewasanappreciableincreaseintheconcentrationofdaidzein,
glycitein, and genistein from 6 h of incubation and they continued
to increase up to 36 h for L. acidophilus L10, 24 h for B. lactis B94,
and 12 h for L. casei L26. L. acidophilus L10 yielded a 63% increase
in aglycone concentration at 36 h, whereas B. lactis B94 and L. ca-
sei L26 showed 77% and 71% increase in aglycones at 24 and 12
h, of incubation, respectively, compared to nonfermented soymilk
(Figure 1).
Figure 4---β-Gluco-
sidase activity (μmol
per mL) in soymilk
undergoing
fermentation by
L. acidophilus L10, B.
lactis B94, and L.
casei L26 for 6, 12, 24,
36, and 48 h at 37◦C
as single cultures.
L10 = L. acidophilus,
B94 = B. lactis, L26 =
L. casei; (error bars
represent a pooled
standard error of the
mean; n = 6; SEM =
0.01 μmol/mL).
Discussion
A
biotic organisms. Previous studies have shown that these bacte-
ria produce α-galactosidase required to metabolize raffinose and
stachyose and grow well in soymilk (Scalabrini and others 1998;
Donkor and others 2006). The growth of the organisms declined af-
ter the respective peak growth period, possibly due to diminishing
nutrient supply in the medium. A similar trend of cell population
decline was observed during storage at 4◦C for 28 d.
During soymilk fermentation, the main metabolic end prod-
ucts include organic acids. These lead to a reduction in the pH of
soymilk. Previous studies have reported similar findings (Angeles
and Marth 1971; Kamaly 1997; Liu 1997). Acetic acid is an unde-
sirable end product in fermented soymilk due to its “vinegary” fla-
vor. Therefore the high production of lactic acid over acetic acid
by lactobacilli and bifidobacteria in this study was desirable for
the production of fermented soymilk. Similar results were reported
by Donkor and others (2005) in soy yogurt made with commercial
soymilk and fermented by L. acidophilus, Bifidobacterium, and L.
casei and during storage at 4◦C.
The respective maximum β-glucosidase activities observed for
each culture showed that the period of fermentation at which the
enzyme activity was highest corresponded to their cell popula-
tions. The increased β-glucosidase activity between 24 and 36 h
was significant (P < 0.05) for L. acidophilus L10, which had the
highest β-glucosidase activity compared to B. lactis B94 and L. ca-
sei L26. Otieno and others (2006) also reported that L. acidophilus
showed the highest β-glucosidase activity at 24 h of fermentation
in soymilk compared to Bifidobacterium spp. and L. casei. Gener-
ally, β-glucosidase activity was found to be strain (P < 0.0436) and
time (P < 0.0001) dependent and, therefore, the enzyme activity
varied among the organisms used during the 48 h fermentation
at 37◦C and this pattern continued throughout storage at 4oC. L.
acidophilus, Bifidobacterium, and L. casei were reported to show
strain-dependent β-glucosidase activity in soymilk (Tochikura and
others 1986; Otieno and others 2005). The β-glucosidase enzymes
produced by these microoganisms are responsible for the break-
down of β-1-6 glucosidic bond, which conjugates the pran ring of
isoflavone and the sugar moieties. These enzyme activities signifi-
cantly increased the content of isoflavone aglycones in fermented
soymilk after incubation with L. acidophilus, Bifidobacterium, and
ll cultures showed good growth characteristics in soymilk, in-
dicating that soymilk was a good delivery medium for pro-
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