Food Chemistry 114 (2009) 829–835

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Selenium-containing green tea has higher antioxidant and prebiotic activities

than regular green tea
A.L. Molan a,*, J. Flanagan b,1, W. Wei c, P.J. Moughan b
a
Institute of Food, Nutrition and Human Health, Massey University, Private Bag 11 222, Palmerston North 4442, New Zealand
b
Riddet Institute, Massey University, New Zealand
c
Roslin Institute (Edinburgh), Roslin, Midlothian, EH25 9PS, Scotland, UK

a r t i c l e i n f o a b s t r a c t

Article history: The effects of selenium-containing green tea (SGT; 1.4 mg selenium/kg) and China green tea (CGT;
Received 5 August 2008 0.13 mg selenium/kg) on the in vitro growth of lactobacilli and bifidobacteria were investigated using
Received in revised form 4 September 2008 pure and mixed cultures. SGT had significantly higher phenolic contents (TPC), higher reducing activity,
Accepted 14 October 2008
higher DPPH free-radical scavenging activity, and higher ferrous-ion chelating activity (P < 0.05–0.0001)
than CGT. The addition of aqueous extracts from CGT to Mann-Rogosa-Sharpe (MRS) broth at 10% and
25% (v/v) resulted in small but nonsignificant (P > 0.05) increases in the numbers of Lactobacillus rhamno-
Keywords:
sus and Bifidobacterium breve over the control incubations (without tea). Addition of 10% and 25% of SGT
Green tea
Selenium
extract resulted in a significant increase (P < 0.05–0.0001) in the number of lactobacilli and bifidobacteria
Prebiotic activity recovered from batch fermentation while CGT did not increase the number of bifidobacteria. The higher
Antioxidant activity prebiotic activity of SGT over CGT may be related to the higher TPC or minerals, notably selenium or a
combination of these factors. To test whether selenium itself has an effect on bacterial growth, Na-sele-
nite and Na-selenate were added alone or in combination with CGT to the MRS broth containing pure cul-
ture of L. rhamnosus. Growth of this bacterium was enhanced relative to the control incubation of MRS
only. When added in combination with CGT, Na-selenate was more effective at enhancing the growth
of L. rhamnosus than Na-selenite. The prebiotic effect of SGT could be largely explained by its selenium
content.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction food products. Although probiotic bacteria can survive transit of

the gastrointestinal tract, some studies have shown that they do
Probiotics have been defined as bacterial preparations that im- not colonize and grow during short or even after prolonged feeding
pact clinically verified beneficial effects on the health of the host periods (Klaenhammer, 2000; Tannock et al., 2000). As a conse-
when consumed orally (Salminen, Gueimonde, & Isolauri, 2005). quence, there is a need for complementary strategies that promote
It is now generally accepted that the composition of the human growth of these desirable lactic acid bacteria in the colon.
intestinal microbiota has an important role in maintaining optimal Prebiotics are dietary factors especially intended to stimulate
health and preventing chronic diseases. The use of probiotic bacte- the growth of probiotic bacteria, and bifidobacteria in particular
ria for the prevention and treatment of intestinal disorders has (Grizard & Barthomeuf, 1999). For example, inulin has been shown
been suggested for many years (Rolfe, 2000). Areas of health in several well-designed in vitro studies to stimulate the growth of
improvement due to probiotic consumption range from combating bifidobacteria (Kaplan & Hutkins, 2000; Rao, 1999). Moreover, it
gut inflammatory diseases, to the prevention/alleviation of aller- has been demonstrated that dietary consumption of non-digestible
gies and increased protection against infectious (viruses, bacteria, oligosaccharides has a positive impact on the numbers of bifido-
fungi) as well as non-infectious (e.g. cancer) diseases (reviewed bacteria in human gut (Bouhnik et al., 1997; Gopal, Prasad, & Gill,
by Cross, 2002). Accordingly, modification of the human intestinal 2003). The effects of carbohydrate-type prebiotics may not always
microbiota to incorporate probiotic (exogenous) species has be- be beneficial, as they can also encourage the growth of non-probi-
come an important objective in nutritional science, and food man- otic bacteria. Some studies have demonstrated that the use of fruc-
ufacturers have responded by developing a range of functional to-oligosaccharides (FOS) resulted in enhanced growth of
Eubacterium biforme and Clostridium perfringens (Bello, Walter,
* Corresponding author. Tel.: +64 06 350 4799; fax: +64 06 350 4665.
Hertel, & Hammes, 2001). Accordingly, new alternative non-carbo-
E-mail address: [email protected] (A.L. Molan). hydrate prebiotics to stimulate the growth of probiotic bacteria
1
Present address: Glanbia Nutritionals, Kilkenny, Ireland. without unwanted side effects are needed.

0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2008.10.028
830 A.L. Molan et al. / Food Chemistry 114 (2009) 829–835

Green tea is a source of many essential dietary compounds and reader. The working FRAP reagent was prepared by mixing 10 vol-
trace elements for human health (Cabrera, Artacho, & Gimenez, umes of 300 mmol/l acetate buffer, pH 3.6, with 1 volume of
2006). In this study we show that green tea generated from tea 10 mmol/l TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mmol/l hydro-
trees grown in certain areas of China is additionally enriched in chloric acid and with 1 volume of 20 mmol/l ferric chloride. A
several minerals and particularly selenium. Selenium is an impor- standard curve was prepared using different concentrations
tant trace mineral and its deficiency seriously affects the health of (200–2000 lmol/l) of FeSO4  7H2O. The antioxidant capacity
both humans and animals (Diaz-Alarcon, Navarro-Alrcon, & based on the ability to reduce ferric ions of the extract was
Serrana, 1994; Levander, 1987). expressed as micromole FeSO4 equivalents per litre of aqueous
The objective of this study was to evaluate the antioxidant extracts. All solutions were used on the day of preparation and
activity of a specific green tea (containing selenium) extract and all determinations were performed in triplicate.
its potential to act as a prebiotic to enhance the viability and
growth of beneficial lactic acid bacteria using both pure and mixed 2.5. Scavenging of diphenyl-picrylhydrazyl (DPPH) radicals
bacterial cultures under in vitro conditions. In addition, the effects
of sodium (Na) selenite and Na-selenate on the growth of Lactoba- This assay detects scavenging of free radicals by the tested com-
cillus rhamnosus were investigated when added alone or in combi- pound through the scavenging activity of the stable 2, 2-diphenyl-
nation with CGT. The later was undertaken to evaluate the effect of 1-picrylhydrazyl (DPPH) free radical. This assay was performed
selenium alone versus tea enriched with selenium. using a previously described method (van Amsterdam et al.,
1992) with some minor modifications. Briefly, 25 ll of tea extract
was allowed to react with 250 ll of 0.2 mM DPPH in 95% ethanol
2. Materials and methods in a 96-well microplate. The plate was then incubated at 37 °C
for 30 min after which the absorbance was measured at 550 nm
2.1. Preparation of tea extracts using a microplate reader. Scavenging capacity of the sample was
compared to that of ascorbic acid as a positive control (0.1–
China green tea (CGT) was purchased from a retail shop in New 1.0 mM ascorbic acid).
Zealand while selenium-containing green tea (SGT) was obtained The antiradical activity was calculated as a percentage of DPPH
from China. SGT is not presently available on the market. The aque- decolouration relative to a negative control using the following
ous extracts were made by adding 100 ml water (100 °C) to 1 g (1% equation:
extract) or 2 g (2% extract) tea leaves and brewing for 10 min with
Antiradical activityð%Þ
stirring and removing solid matter by filtration. The extracts were
ðabsorbance of control incubation  absorbance of the tea extractÞ
filter-sterilized before being added to the MRS broth. Each experi- ¼ :
ment was run in duplicate and the whole experiment was repeated absorbance of control incubation  100
to confirm the results.

2.2. Mineral analysis of dry tea material 2.6. Ferrous-ion chelating assay

Dry leaves of both SGT and CGT were ground into fine powders The ferrous-ion chelating assay (FCA) used by Singh and Rajini
and sent to the New Zealand Laboratory Services, Ruakura (2004) and Chan, Lim, and Chew (2007) was followed with slight
Research Centre, Hamilton, New Zealand for mineral analysis. modifications. Solutions of 2 mM FeSO4 and 5 mM ferrozine were
The Ca, Fe, K, Na and P were determined by inductively coupled prepared. Diluted FeSO4 (100 ll) was mixed with 100 ll of sample,
plasma-optical emission spectrometry (ICP-OES), while Cu, Mn, followed by 100 ll of diluted ferrozine. Assay mixtures were al-
Se and Zn were determined by inductively coupled plasma-mass lowed to equilibrate for 10 min before measuring the absorbance
spectrometry (ICP-MS). at 560 nm using a plate reader. Tea extracts were assayed in dupli-
cate. The ability of the tea extracts to chelate ferrous ions was cal-
2.3. Total polyphenols contents (TPC) culated relative to a negative control using the equation:
Chelating activity%
The amount of total phenolic content (TPC) in tea extracts was
ðabsorbance of control incubation  absorbance of the tea extractÞ
determined according to the Folin-Ciocalteu procedure as used by ¼ :
absorbance of control incubation  100
Molan, Lila, and Mawson (2008a) with some modifications. Briefly,
an aliquot of 12.5 ll of water-soluble extract was mixed with
250 ll of 2% sodium carbonate solution in 96-well microplates
and allowed to react for 5 min at room temperature. Then, 2.7. In vitro assay to monitor the prebiotic activity using pure cultures
12.5 ll of Folin-Ciocalteu phenol reagent (50%) was added and al- of lactic acid bacteria
lowed to stand for 30 min at room temperature before the absor-
bance of the reaction mixture was read at 650 nm using a plate A series of in vitro experiments were conducted to investigate
reader. Calibration was achieved with an aqueous gallic acid solu- the impact of two tea extracts on the growth of two strains of lactic
tion (100–1000 lg/ml). The TPC of the extract was expressed as mg acid bacteria. Pure cultures of Lactobacillus rhamnosus and Bifido-
gallic acid equivalent (GAE) per gram of tea leaves on a dry basis. bacterium breve were obtained from the culture collection held
by Environmental Science and Research (ESR), New Zealand. These
2.4. Ferric reducing antioxidant power assay strains were grown to stationary phase in Mann-Rogosa-Sharpe
(MRS) medium and then 1% (v/v) inoculums from these cultures
The capacity to reduce ferric ions was determined using the Fer- were introduced into 2 ml of fresh MRS broth containing different
ric Reducing Antioxidant power (FRAP) assay as described by concentrations of the tea extracts. The extracts were added to MRS
Benzie and Strain (1996). Briefly, an aliquot of 8.5 ll of tea (1% or broths at 10% and 25% (v/v). The tubes were incubated at 37 °C for
2% water extracts) was added to 275 ll of diluted FRAP reagent 48 h and at the end of the incubation period, the broths from con-
using a microplate and the plates were incubated at 37 °C for trol and extract-containing incubations were serially diluted 10-
30 min before measuring the absorbance at 395 nm using a plate fold in fresh MRS broth and then 100 ll aliquot of each dilution
 A.L. Molan et al. / Food Chemistry 114 (2009) 829–835 831

was spread in duplicate on the surface of the plates containing MRS ormaldheyde in PBS (pH 7.2) overnight at 4 °C. Fixed samples were
agar, for enumeration of L. rhamnosus. For B. breve, MRS broth was washed in PBS and stored in a known volume of 50% (v/v) ethanol-
supplemented with 0.05% cysteine hydrochloride (MRSc). The MRS PBS at 20 °C until time of hybridization. Aliquots (5 ll) of fixed
agar plates were incubated anaerobically at 37 °C for 48 h and then bacterial cells were applied to Teflon-coated microscopic slides
bacteria enumerated. Growth of the strains was monitored by (BIOLAB, New Zealand; 10 wells) and air dried. The bacterial cells
counting the viable bacterial cells using a plate reader. Controls were then dehydrated with a series of solutions containing 50%,
included a positive control (medium + bacteria) and a negative 80%, and 99.5% ethanol (3 min for each concentration). The bacte-
control (medium only) containing 10 and 25% sterile distilled rial cells fixed on the glass slides were hybridized by addition of
water. 8 ll of hybridization buffer (0.9 M NaCl, 0.01% sodium dodecyl sul-
fate, 20 mM Tris-HCl, 20% deionized formamide; pH 7.2) with
2.8. Effects of Sodium selenite and sodium selenate on growth of L. 0.5 ll of Cy3-labeled oligonucleotide specific probes (50 ng/ll).
rhamnosus using pure culture The slides were hybridized at 46 °C for 2 h in a plastic box contain-
ing wet sponges (soaked in hybridization buffer). After hybridiza-
Filter-sterilized aqueous solutions of sodium (Na) selenate and tion, the slides were rinsed with warm hybridization buffer at
Na-selenite (Sigma–Aldrich, Australia) were added to selenium- 48 °C and washed in pre-warmed washing buffer (225 mM NaCl,
free MRS broth to a final volume of 4 ml in sterilized Hungate 0.01% sodium dodecyl sulfate, 20 mM Tris-HCl; pH 7.2) for
tubes. A total of 4 concentration treatments plus a control treat- 20 min at 48 °C. After the washing step, the slides were rinsed with
ment were performed in duplicates, and the nominal concentra- ice-cold distilled water and thoroughly dried before being ob-
tions used were 1, 2, 3, 4 and 5 lg selenium/ml in selenate or served using a fluorescence scanning microscope.
selenite forms. The growth and viability of bacteria were evaluated The dried slides were examined with an Olympus BX51 micro-
by counting the living cells using MRS agar media as described scope, under 400X magnification. The images were captured using
previously. an Optronics MagnaFIRE SS99802 digital camera with MagnaFIRE
frame-grabbing software on a Pentium IV computer (Manawatu
2.9. Testing for prebiotic effects of extracts with faecal batch-culture Microscopy and Imaging Centre, Massey University, Palmerston
fermentation North, New Zealand). Fluorescent cells were counted automatically
in 5 randomly selected fields/slide using ImageJ (Abramoff,
A small scale in vitro fermentation method was used to study Magelhaes, & Ram, 2004). The probes used in this study were Bif
the growth of faecal bacteria in response to the tea extracts. Ex- 164 with a sequence of 5/-CATCCGGCATTACCACCC-3 and Lac 158
tracts, 10% and 25% (v/v), were tested in this system. The medium with a sequence of 5-GGTATTAGCAYCTTCCA-3.
contained (per liter) 2 g of peptone water, 2 g of yeast extract, 0.1 g
of NaCl, 0.04 g of K2HPO4, 0.01 g of MgSO4  7H2O, 0.01 g of 2.11. Statistical analysis
CaCl2  6H2O, 2 g of NaHCO3, 0.005 g of haemin, 0.5 g of L-cysteine
HCl, 0.5 g of bile salts, 2 ml of Tween 80, 10 ll of vitamin K, and Colony populations for each bacterial group were expressed as
4 ml of 0.025% (w/v) resazurin solution. Batch cultures were set log colony forming units (cfu) or log number/ml medium. Logarith-
up in 15 ml sterile tubes containing 4 ml pre-reduced sterile med- mically-transformed data were analysed by one way analysis of
ium. Human faecal samples were collected from healthy volun- variance using SAS (version 9.1) with the level of significance set
teers, who had not used antibiotics for the last three months at P < 0.05.
prior to the sampling date, and had no recent history of gastroin-
testinal complaints. Three volunteers were asked to defecate in 3. Results
plastic bags and immediately the samples were mixed and homog-
enized by kneading the bag and a subsample of about 10 g was put 3.1. Mineral contents
in a pre-weighed glass container with 90 ml anaerobic buffered
peptone water with 0.5 g/l cysteine-HCl. Each batch culture was The SGT and CGT powdered leaves were analyzed for mineral
inoculated with 40 ll of the faecal slurry, to give a final concentra- content (Table 1). Elemental analysis indicated that the SGT leaves
tion of 0.1% (v/v). All additions and inoculations were carried out had a ten-fold higher mean total selenium concentration (1.4
inside an anaerobic cabinet. Each fermentation experiment was versus 0.13 mg of Se/kg; P < 0.0001). Moreover, SGT contains
carried out in duplicate at an incubation temperature of 37 °C. higher concentrations of phosphorus, potassium, iron, zinc and
One sample was prepared, as a control, without any extract copper than CGT (Table 1), but CGT contains more calcium and
addition. Batch cultures were incubated anaerobically at 37 °C for manganese.
48 h.
At the end of incubation, serial dilutions (from 103 to 106)
from each incubation were made in sterile-filtered phosphate buf-
fer (PBS; pH 7.2) and the numbers of bifidobacteria and lactobacilli
Table 1
were counted using the fluorescent in situ hybridization (FISH) Mean (± SEM) mineral contents of selenium green tea (SGT) and China green tea
method. (CGT).

Mineral Unit Concentration

2.10. Fluorescence in situ hybridization analysis of microbiota in faecal
SGT CGT
batch-culture fermentation
Phosphorus g/100 g 0.46 ± 0.083 0.25 ± 0.005
The probes used in the study were Bif164 and Lab158, specific Potassium g/100 g 1.78 ± 0.055 1.5 ± 0.085
Calcium g/100 g 0.33 ± 0.077 0.49 ± 0.015
for bifidobacteria and lactobacilli, respectively. These were com- Sodium g/100 g <0.02 <0.02
mercially synthesized and labeled with the fluorescent dye Cy3 Iron mg/kg 407 ± 13.0 305.0 ± 64
(GeneWorks, Australia). The procedure described by Dinoto et al. Manganese mg/kg 1159 ± 140.5 1619 ± 428.0
(2006) was followed with some modifications. In short, FBCF mix- Zinc mg/kg 49.1 ± 11.1 43.5 ± 11.5
Copper mg/kg 25.5 ± 2.1 16.0 ± 3.8
tures were centrifuged at low speed (700 g) to remove debris, and
Selenium mg/kg 1.4 ± 0.075 0.13 ± 0.002
the bacteria containing supernatant was fixed in 4% (w/v) paraf-
832 A.L. Molan et al. / Food Chemistry 114 (2009) 829–835

Table 2 3.3. Prebiotic activities with pure cultures

Mean antioxidant activity [ferric reducing antioxidant power (FRAP; lmol/l); DPPH
scavenging activity and ferrous-ion chelating activity (FCA)] and total phenolic
contents (TPC; mg GAE/g dry leaves) of China green tea (CGT) and selenium-
Addition of aqueous extracts from CGT to MRS broth at 10% (v/
containing tea (SGT). v) resulted in slight (but not statistically significant) increases in
the numbers of L. rhamnosus over the control incubations (without
CGT SGT Statistical significance
tea) (Fig. 1A). With a 25% addition, CGT enhanced growth (Fig. 1b)
TPC (mg/g dry leaves) with the difference approaching significance (P = 0.07). On the
1% extract 34.1 ± 4.2 60.5 ± 2.96 P < 0.0001
2% extract 55.1 ± 1.6 94.2 ± 1.5 P < 0.0001
other hand, addition of 10% and 25% of SGT (Figs. 1A,B) resulted
in significant (P = 0.0172 for 10% and P = 0.0073 for 25%) increases
FRAP (lmol/l)
1% extract 9002.8 ± 60.6 9947.6 ± 37 P < 0.001
in the population sizes of L. rhamnosus as compared to the control
2% extract 10186.3 ± 135.5 11132.7 ± 208 P < 0.001 incubations.
DPPH (%)
Addition of water extract from CGT to MRS broth at either 10 or
1% extract 69.4 ± 0.76 71.5 ± 0.6 P < 0.05 25% additions did not promote growth of B. breve although the dif-
2% extract 72.4 ± 0.18 74.2 ± 0.85 P < 0.05 ference at 25% approached significance (P = 0.057; Fig. 2B). Fig. 2
FCA (%) clearly shows that addition of 10% and 25% of SGT extract to the
1% extract 34.04 ± 0.57 39.3 ± 1.3 P < 0.05 incubations containing B. breve resulted in a significant increase
2% extract 40.2 ± 1.4 46.8 ± 1.1 P < 0.05 (P = 0.0016 for 10% and P = 0.0009 for 25%) in the numbers of this
Means of duplicate incubations for two separate experiments. Statistical analysis bacterium over the control incubations (without tea).
based on ANOVA (compared to values in the same row).
3.4. Testing for prebiotic effects with faecal batch fermentation
(in vitro fermentation method)

3.2. Total polyphenol contents and antioxidant activities Addition of 10% and 25% extracts from the SGT resulted in sig-
nificant increases (P = 0.0116 and P = 0.0002, respectively) in the
SGT had significantly higher (P < 0.0001) TPC than CGT at both number of lactobacilli recovered from the faecal batch cultures as
1% and 2% extractions (Table 2). Aqueous extracts from SGT also assessed by the FISH method (Table 3). Addition (10%) of CGT to
showed significantly higher reducing activity as measured by FRAP the faecal batch cultures did not lead to an increase in the number
(P < 0.01), higher free radical scavenging activity as measured by of lactobacilli to a statistically significant extent, while addition of
DPPH assay (P < 0.05) and higher ferrous-ion chelating activity 25% of the same tea resulted in a significant (P = 0.0225) increase in
(P < 0.05–0.01) than CGT. the numbers of lactobacilli over the control incubations.

A 10
Log number of Lactobacillus

Log number of bifidobacterium

* A 10
rhamnosus (CFU/ml)

9
breve (CFU/ml)

9 *

8
8

7
7
Control CGT SGT Control CGT SGT

B 10
Log number of Lactobacillus

Log number of bifidobacterium

** B 10
rhamnosus (CFU/ml)

breve (CFU/ml)

9
9 **

8
8

7 7
Control CGT SGT Control CGT SGT
Fig. 1. Effect of water extracts from China green tea (CGT) and natural selenium- Fig. 2. Effect of water extracts from China green tea (CGT) and natural selenium-
containing green tea (SGT) on the survival of Lactobacillus rhamnosus NZRM 299 containing green tea (SGT) on the survival of Bifidobacterium breve NZRM 3932
(Mean ± standard error). This bacterium was incubated in MRS medium containing (Mean ± standard error). This bacterium was incubated in MRS medium containing
different concentrations (10%; A and 25%; B) of these teas. Statistical analysis based different concentrations (10%; A and 25%; B) of these teas. Statistical analysis based
on ANOVA, *: P < 0.05 and **: P < 0.01 compared with the control group. on ANOVA, *: P < 0.05 and **: P < 0.01 compared with the control group.
 A.L. Molan et al. / Food Chemistry 114 (2009) 829–835 833

Table 3 rhamnosus at the highest concentration (5 lg/ml; Fig. 4) only.

Mean effects of selenium-containing green tea (SGT) and China green tea (CGT) Contrary to Na-selenite, Na-selenate was able to enhance growth
extracts on the population size (log number per ml ± standard error) of lactobacilli
and bifidobacteria in a faecal batch culture fermentation system after 24 h incubation
significantly when added to MRS broth containing 10% water-
at 37 °C. soluble extract from CGT at all concentrations tested (Fig. 4).
Gross examination of the data presented in Figs. 3 and 4 pro-
Concentration CGT SGT
(mg/ml)
duces some evidence that the effect of sodium selenite (see
Lactobacilli Bifidobacteria Lactobacilli Bifidobacteria Fig. 3) may have been nullified by CGT.
0 (control) 7.92 ± 0.07 7.32 ± 0.01 7.92 ± 0.077 32 ± 0.01
10% 7.98 ± 0.05 7.54 ± 0.05 8.25 ± 0.09* 7.84 ± 0.15*
4. Discussion
25% 8.20 ± 0.04* 7.72 ± 0.07 8.77 ± 0.04*** 8.17 ± 0.19**

Means of duplicate incubations for two identical experiments. The principal finding of this study is that water extracts pre-
*
P < 0.05 (compared to control incubation in the same column).
** pared from green tea containing high concentrations of organic
P < 0.01 (compared to control incubation in the same column).
***
P < 0.0001 (compared to control incubation in the same column). selenium are more effective at promoting the growth of pure cul-
tures of Lactobacillus rhamnosus and Bifidobacterium breve than
green tea containing normal levels of selenium. Further, there
Addition of 10% and 25% extracts from the SGT resulted in sig- was some evidence for a prebiotic effect of green tea containing
nificant increases (P = 0.0222 and P = 0.0032) in the number of bif- normal levels of selenium. The extracts from the SGT were also
idobacteria recovered from the faecal batch cultures (Table 3). able to promote the growth of faecal lactobacilli and bifidobacteria
Addition (10%) of CGT to the faecal batch cultures did not increase in the faecal batch fermentation system especially at high concen-
the number of lactobacilli to a significant extent while addition of trations and showed significant superiority over CGT. This system
25% of the same tea resulted in a border-line significant is a simple but very efficient culturing model for assessing the ef-
(P = 0.0555) increase in the numbers of bifidobacteria over the con- fect of test compounds on the growth of faecal bacteria using fresh
trol incubations. human faeces as an inoculum (Sanz et al., 2005). Moreover, it has
been shown that the results obtained with such techniques, at
3.5. Effects of Na-selenite and Na-selenate on growth of L. rhamnosus least for inulin and fructooligosaccharides, are consistent with
using a pure culture those from human studies (Gibson, Beaty, Wang, & Cummings,
1995).
In order to test the hypothesis that selenium itself may have an Although the mechanism by which tea extracts increased the
effect on bacterial growth, the primary inorganic forms of sele- growth of L. rhamnosus and B. breve in this study is not known, a
nium, Na-selenite and Na-selenate were added alone or in combi- possible partial explanation for this enhancing effect is the ability
nation with CGT to the MRS broth containing viable cells of L. of polyphenols in green tea, to act as antioxidant and anti-
rhamnosus to monitor their impact on the growth of this bacterium radical agents (Molan, De, & Meagher, 2008b; Serafini, Ghiselli, &
in vitro. At all concentrations of Na-selenate tested a significant in- Ferro-Luzzi, 1996; Xu, Yang, Chen, Hu, & Hu, 2003), to modulate
crease (P < 0.05–0.01) in the numbers of viable bacterial cells was the oxidative stress in the medium generated by the metabolic
seen relative to the control incubation containing MRS broth only activities and consequently provide a better environment for the
(Fig. 3). In contrast, Na-selenite was able to promote the growth growth and multiplication of these bacteria. Our results showed
of the same bacterium to a significant extent at 2–5 lg/ml high antioxidant activities for both CGT and SGT, but with SGT
(P < 0.05–0.01), but not at a concentration of 1 mg/ml. CGT on its having significantly greater antioxidant potential. Furthermore,
own did not increase growth of L. rhmnosus (Figs. 3 and 4), whereas our study shows, for the first time, that inorganic forms of
SGT had a similar effect to added Na-selenate. selenium (Na-selenate and Na-selenite) have a prebiotic activity
Addition of Na-selenite to MRS containing 10% (v/v) water-sol- as evidenced by their ability to promote the growth of L. rhamnosus
uble extract from CGT (1% extract) enhanced the growth of L. and B. breve under in vitro conditions. Indeed it appears that

10

8 * * * ** * * ** ** ** **
Log CFU/ml

0
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l)

l)

l)

T
l)

l)

l)

l)

T
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/m

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G
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C
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on

ug

ug

ug
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ug

ug
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C

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te

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te
te

te
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na

na
na
na
le

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le

le

le
le

le

le
Se

Se

Se

Se

Se

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Se

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Fig. 3. Effect of sodium (Na) selenite and Na-selenate on the growth and viability of Lactobacillus rhamnosus when added alone to MRS broth (Mean ± standard error).
Statistical analysis based on ANOVA, *: P < 0.05 and **: P < 0.01 compared with the control group.
834 A.L. Molan et al. / Food Chemistry 114 (2009) 829–835

10
* ** ** ** * *
Log CFU/ml 8

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te

te

te

e
te

te

t
ni

ni

ni

ni

na

na

na

na
le

le

le

le

le

le

le

le
Se

Se

Se
Se

Se

Se

Se

Se
Fig. 4. Effect of sodium (Na) selenite and Na-selenate on the growth and viability of Lactobacillus rhamnosus when added in combination with China green tea (CGT) to MRS
broth. Statistical analysis based on ANOVA, *: P < 0.05 and **: P < 0.01 compared with the control group.

Na-selenate was as effective as SGT in enhancing bacterial growth by lowering pH. It was reported that although the catechins were
and the pronounced effects of SGT observed here may be due to its bactericidal, there was no effect on lactic acid bacteria.
high selenium content. It also appears that Na-selenate may In a further study, Goto et al. (1998) administered 300 mg of
behave differently to Na-selenite, in this respect. catechin per day to 15 subjects over a 3-week period. They found
Recently, Arauz et al. (2007) studied the protective role of sele- significantly increased faecal levels of lactobacilli and bifidobacte-
nium against cadmium (Cd) toxicity in Lactobacillus casei rhamno- ria while levels of Enterobacteriaceae, Bacteroidaceae and eubacte-
sus bacteria and found that the growth inhibition was observed ria decreased significantly. Ishihara, Chu, Akachi, and Juneja (2001)
in all cultures exposed to Cd alone while in the presence of Na- evaluated extracts from two green teas for their effects in inhibit-
selenite and Cd, bacterial growth and cell viability were improved ing the growth of pathogenic bacteria isolated from farm animals
as compared to bacteria exposed only to Cd. However, the same and their effects on improving intestinal microflora balance in
researchers found that the presence of 1 mg/l Na-selenite in the calves. The authors reported that feed containing green tea extracts
medium did not affect culture growth. The ability of lactic acid bac- maintained high faecal counts of Bifidobacterium species and Lacto-
teria to incorporate selenium from the growth medium has been bacillus species in calves.
demonstrated (Arauz et al., 2007; Calomme, Hu, Vanden Branden, The results of the present study clearly show that the high-sele-
& Vanden Berghe, 1995). nium tea (SGT) had a significantly higher total phenolic content and
From a health perspective, the present results are important a higher antioxidant activity, as measured by FRAP, DPPH and FCA
due to the growing interest in probiotic bacteria and the perceived assays, than CGT which contained a normal amount of selenium.
benefit of increasing their numbers in the gastrointestinal tract. There were other differences between the two teas, however, so
Probiotic bacteria may enhance gut health by competitively we cannot ascribe the difference in antioxidant activity to selenium
excluding pathogens from the occupation of adhesion sites or by with certainty. Other studies have demonstrated that polyphenolic
influencing the gut environment via the secretion of simple or compounds in green tea extracts are potent free-radical scavengers
complex molecules. Increasing the numbers of lactic acid bacteria due to their abilities to act as hydrogen or electron donors (Chan
(lactobacilli and bifidobacteria) in the colon was found to reduce et al., 2007; Higdon & Frei, 2003). In one study, the effect of foliar
the formation of ammonia, skatole, harmful amines and other pro- application of selenium on increasing the antioxidant activity of
carcinogens in the large intestine and the carcinogenic load on the tea was investigated (Xu, Yang, Chen, Hu, & Hu, 2003) and the re-
intestine (Burns & Rowland, 2000; Yamamoto, Juneja, Chu, & Kim, sults showed that the radical scavenging ability of the tea extracts
1997). Probiotics and prebiotics, which modify the microflora by were in the following order: selenium-enriched tea obtained by fer-
increasing numbers of lactobacilli and/or bifidobacteria in the co- tilization with selenate, selenium -enriched tea obtained by fertil-
lon, have been a particular focus of attention in this regard (Burns ization with selenite and then regular green tea.
& Rowland, 2000).
While the anti-bacterial effects of green tea are relatively well
known (Hara-Kudo et al., 2005), few studies have shown that green 5. Conclusions
tea and its polyphenols can promote the growth of beneficial pro-
biotic bacteria. Hara et al. (1995) examined the effect of tea poly- Water extracts prepared from SGT have significantly higher
phenols on the fecal flora and metabolites of pigs. This study antioxidant activity and are more effective at promoting the
involved feeding eight 30-day old pigs a diet supplemented with growth of defined (pure cultures) or undefined (mixed cultures)
0.2% tea polyphenols for 2 weeks. The authors reported a signifi- cultures of lactobacilli and bifidobacteria than CGT. The higher
cant increase in the numbers of lactobacilli and a significant de- TPC and higher concentration of organic selenium in SGT or the
crease in total bacteria and bacteroidaceae counts. Hara (1997) combined effects of both may underlie this superiority of SGT over
conducted a human trial showing that the administration of CGT. It would appear that the difference in the amount of selenium
100 mg of tea catechins three times daily with meals for three had a major effect. The chemical identity of the selenium in SGT re-
weeks decreased putrefactive products and increased organic acids mains unknown. Further studies are needed to identify the form of
 A.L. Molan et al. / Food Chemistry 114 (2009) 829–835 835

selenium found in the SGT and to determine its bioavailability and Goto, K., Kanaya, S., Nishikawa, T., Hara, H., Terada, A., Ishigami, T., & Hara, Y. (1998).
The influence of tea catechins on fecal flora of elderly residents in long-term
biological activities. SGT shows promise as a prebiotic material.
care facilities. Annals of Long-Term Care, 6, 43–48.
The present work is in vitro based and it is now necessary to con- Grizard, D., & Barthomeuf, C. (1999). Non-digestible oligosaccharides used as
firm the findings in vivo. This will be the objective of a further prebiotic agents mode of production and beneficial effects on animal and
study by our group. human health. Reproduction, 39, 563–588.
Hara, Y. (1997). Influence of tea catechins on the digestive tract. Journal of Cellular
Biochemistry, 27, 52–58.
Acknowledgment Hara, H., Orita, N., Hatano, S., Ichikawa, H., Hara, Y., Matsumoto, N., Kimura, Y.,
Terada, A., & Mitsuoka, T. (1995). Effect of tea polyphenols on fecal flora and
fecal metabolic products of pigs. Journal of Veterinary Medical Science, 57,
Part of this work was financially supported by the DairyGold 45–49.
Cooperative Society, Ireland. We thank Shampa De for technical Hara-Kudo, Y., Yamasaki, A., Sasaki, M., Okubo, T., Minai, Y., Haga, M., Kondo, K., &
assistance. Sugita-Konishi, Y. (2005). Antibacterial action on pathogenic bacterial spore
by green tea catechins. Journal of the Science of Food and Agriculture, 85,
2354–2361.
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