Eur Food Res Technol (2009) 229:153–157
DOI 10.1007/s00217-009-1036-x
ORIGINAL PAPER
EVect of lactobacillus fermented beetroot juice on composition
and activity of cecal microXora of rats
Elrbieta Klewicka · Zenon Zdujczyk · Jerzy Jumkiewicz
Received: 5 September 2008 / Revised: 5 February 2009 / Accepted: 16 February 2009 / Published online: 6 March 2009
© Springer-Verlag 2009
Abstract This study aimed at determination of the eVect
caused by ingestion of beetroot juice fermented by
Lactobacillus casei 0920 and Lactobacillus brevis 0944
strains on the state of cecal ecosystem of experimental rats.
The intake of fermented beetroot juice containing 3.5–
4.0 £ 109 CFU/mL live Lactobacillus sp. cells positively
modulated the cecal microXora of the rats and its metabolic
activity. The counts of Lactobacillus sp., BiWdobacterium
sp., Bacteriodes sp., and Enterococcus sp. were maintained
at the level of 8.2–8.6, 6.2–7.5, 8.0–8.3, and 7.3–7.7 log
units, respectively, while the number of Clostridium sp.
cells was increased by 1.1–1.6 log units and Enterobacteri-
aceae bacteria were reduced by 0.8–2.1 log units. In this
study, the selected cecal enzymes such as
-glucosidase,

-glucuronidase, and
-galactosidase as well as the proWle
and concentration of short chain fatty acids (SCFA) were
the biochemical markers of metabolic activity of the intesti-
nal ecosystem. The considerable decrease in activities of

-glucosidase and
-glucuronidase was observed in all three
experimental groups fed with the fermented beetroot juice.
Total concentration of SCFA was the highest (78.1
mol/
100 g BW vs. 59.2
mol/100 g BW in control group) in
intestines of rats fed with 6 mL of fermented beetroot juice
daily. These results prove that the fermented beetroot juice
beneWts cecal microbial activity.
E. Klewicka (&)
Institute of Fermentation Technology and Microbiology,
Technical University of Lodz, 171/173 Wolczanska St.,
90-924 Lodz, Poland
e-mail: klewicka@p.lodz.pl
Z. Zdujczyk · J. Jumkiewicz
Division of Food Science,
Institute of Animal Reproduction and Food Research,
PAS, 10-747 Olsztyn, Poland
Keywords Cecal microbiota · Lactobacillus · Beetroot
juice · Fecal enzyme · SCFA
Introduction
Functional foods have recently gained much attention from
both researches and consumers. This category of food is
expected not only to provide the consumers with nutrients
but also to beneWt consumers’ health in a speciWc manner as
declared by manufacturers. Functional foods comprise
products which reduce blood pressure (low fat milk bever-
ages), probiotics, fruit juices supplemented with calcium,
dietary Wber, prebiotics, energizing beverages, low fat
cheese and bread enriched with macro- and microelements
[1]. Fruit and vegetable juices are particularly promising
raw materials to be used for functional food production
because they contain vitamins, macroelements and (fre-
quently) dietary Wber. Their additional advantage is the
occurrence of natural plant pigments such as carotenoids,
chlorophylls, anthocyanins and betalains, which neutralize
free radicals responsible for processes of degradation of
cellular structures and aberrations of DNA molecules [2–4].
Betanin, which is a natural colorant added (0.1–1% product
weight) to yoghurts, ice creams, sauces, instant drinks and
gelatin-containing desserts, has gained much interest from
food industry. Betacyanins (betanine and isobetanine) rank
among water-soluble red pigments. Their stability is low,
particularly during heating and storage, and therefore lactic
acid fermentation can be an alternative method of preserva-
tion of beetroot juice pressed under high pressure, which is
usually thermally preserved. The disadvantage of the latter
method is a change in beetroot juice coloration from pink-
red to brown. However, the partial recovery of red pig-
ments occurs during lactic acid fermentation due to a drop
123
154
in pH to approximately 4.0 because the beetroot juice
pigments are optimally stable at the latter pH [5]. Initiation
of beetroot juice fermentation with the appropriate starter
culture facilitates production of vegetable preserves with
an enhanced nutritional value and supplementation with
the probiotic microXora (lactic acid bacteria) additionally
improves the health-promoting properties of the products.
The majority of beverages and preparations, which contain
probiotic strains of lactic acid bacteria, are based on milk
and dairy products. Therefore they cannot be consumed by
lactose or milk proteins intolerant humans. Fermented veg-
etable products such as fruit and vegetable juices are an
alternative addressed to this group of consumers. This work
presents the eVects of beetroot juice fermented by Lactobacil-
lus casei 0920 (probiotic strain, according to the guidelines
of FAO/WHO 2001/2002) [6] and Lactobacillus brevis
0944 (plant strain) strains on the state of intestinal ecosys-
tems of experimental animals (rats) and metabolic activity of
their intestinal microXora. The latter was determined on the
basis of
-glucuronidase,
-glucosidase, and
-galactosidase
activities and concentration of short chain fatty acids
(SCFA) in cecal digesta.
Materials and methods
Materials and microorganisms
Beetroot juice derived from the Chrobry variety was used
throughout the studies. The juice was produced under labora-
tory conditions (0.8 L juice from 1 kg beetroots) by using a
juicer, pasteurized (for 10 min at 80 °C) prior to fermentation
and inoculated (10% inoculum, v/v) with selected cultures of
lactic acid bacteria Lactobacillus casei 0920 and Lactobacillus
brevis 0944. The inoculum density was 5 £ 106 CFU/mL.
The fermentation was conducted for 48 h at 30 °C and the
ultimate concentration of Lactobacillus cells was 3.5–
4.0 £ 109 CFU/mL. Chemical composition of fermented
beetroot juice was described by Czyrowska et al. [7].
The strains Lactobacillus casei 0920 and Lactobacillus
brevis 0944 were derived from the Collection of Industrial
Cultures LOCK 105 at the Institute of Fermentation Technol-
ogy and Microbiology, Technical University of Lodz (Poland).
An eVect of the fermented beetroot juice was tested
using model rats of Wistar breed. Three groups of the ani-
mals (8 male specimens in each group) were fed for
4 weeks with the basic casein diet [8] (composition of the
basic diet is shown in Table 1) supplemented with three
diVerent volumes (1.5, 3.0, and 6.0 mL daily) of the fer-
mented beetroot juice. The control group was fed with the
casein diet. The experimental protocol was approved by the
Local Animal Care and Use Committee in Olsztyn (permis-
sion no 53/2007/N).
123
Eur Food Res Technol (2009) 229:153–157
Table 1 Composition of the basic diet
Concentration
(%, dry matter)
Casein 8.0
Soybean protein 8.5
DL-Methionine 0.2
Cellulose 5.0
Soybean oil 8.0
Cholesterol 0.5
Corn starch 63.3
Mineral mix AIN-93G-MX 3.5
Vitamin mix AIN-93G-VM 2.0
From Ref. [8]
Sample collection and preparation
Samples of fresh cecal content were taken from single rats
after the experimental period and immediately placed in an
anaerobic chamber. All preparation procedures were per-
formed in oxygen-free atmosphere (H:N:CO2; 1:8:1). Sam-
ples of approximately 1 g (wet weight) were homogenized
in Wilkins-Chalgren broth (Oxoid) to Wnal ratio 1:10 (w/v).
Then, 1 mL of the cecal homogenate was used to prepare a
series of 10-fold dilutions (from 10¡1 to 10¡10).
Microbial analysis
The cecal microXora of experimental rats was analyzed by
the standard plate tests. The following groups of microor-
ganisms were quantitatively determined on selective media:
Lactobacillus sp., Rogosa Agar (Merck); BiWdobacterium
sp., RB Agar [9]; Clostridium sp., TSC Agar (Merck);
Bacteroides sp., Schaedler Agar (BioMerieux) supple-
mented with 5% (v/v) sheep blood, kanamycine–vankomy-
cine mixture (BioMerieux) and vitamin K 0.01% (w/v);
Enterococcus sp., Bile Aesculin Agar (Merck); Enterobac-
teriaceae family, Mac Conckey Agar (Merck); and the total
number of anaerobic bacteria was determined using Schae-
dler Agar (BioMerieux). Lactobacillus sp. was cultivated
for 48 h at 37 °C in CO2 (5%, v/v) atmosphere (WTC
Binder CO2 Incubator, Germany). The bacteria, BiWdobac-
terium sp., Clostridium sp., Bacteroides sp., and total
anaerobic bacteria, were cultured at 37 °C in the anaerobic
atmosphere of H:N:CO2 (1:8:1) (Anaerobic Workstation
Concept 400, Biotrace Int.) for 4 days.
Biochemical analysis
The activity of microbial enzymes,
-glucuronidase,

-glucosidase, and
-galactosidase, in the cecal digesta was
measured by determining p-nitrophenol released from
Eur Food Res Technol (2009) 229:153–157 155

appropriate p-nitrophenyl-
-D-glycosides [10]. Enzymatic
activity was expressed as micromoles of the product
formed in 1 min (U) per 1 g cecal digesta or protein in the
cecal sample. The protein content in the sample was deter-
mined by Lowry et al. [11] method.
SCFA in fresh cecal contents were determined by gas
chromatography (GC) (Shimadzu GC-14A with a 2.5 £
2.6 mm glass column containing 10% SP-1200/1% H3PO4
on 80/100 Chromosorb WAW; column temp. 110 °C,
detector FID temp. 180 °C; injector temp. 195 °C; Shima-
dzu Corp., Kyoto, Japan). Aliquots of cecal digesta were
mixed with 0.2 mL of formic acid, diluted with deionized
water, and centrifuged at 10000g for 5 min. Samples of the
supernatant were subjected to gas chromatography analysis
[12].
Statistical analysis
The data were analyzed by the Origin Program version 6.1
and with Microsoft Excel version 97. SigniWcance of diVer-
ence between mean values was tested with the Student’s
t-test. A P value of ·0.05 was considered signiWcant.
Results and discussion
The function of lactic acid bacteria strains consists in mod-
ulation and stabilization of intestinal microXora and its met-
abolic activity. In our studies, the beetroot juice was used as
lactic acid bacteria carrier. Because of anticancer activity of
beetroot juice [3] and the occurrence of Lactobacillus sp.
strains, our product possesses two valuable properties of
that characterize functional foods. Furthermore, the product
retains its nutritional value throughout storing in a refriger-
ator for 30 days [13]. Supplementation of the diet of exper-
imental rats with the fermented beetroot juice considerably
reduced the number of Enterobacteriaceae family bacteria
(Table 2). A drop in their number grew with the volume of
ingested juice and approached: ¡0.8 log units for 1.5 mL
(6 £ 109 CFU/dose), ¡1.4 log units for 3.0 mL (1.2 £
1010 CFU/dose), and ¡2.1 log units for 6.0 mL (2.4 £
1010 CFU/dose). Because Enterobacteriaceae strains com-
prise both the typical comensal intestinal microXora, e.g.
Escherichia coli strains and numerous pathogens such as
strains of Salmonella sp., Shigella sp., Proteus sp., and
Klebsiella sp., the decrease in the number of their cells can
be regarded as a beneWcial inXuence of L. casei and L. brevis
strains used for beetroot juice fermentation. This eVect
was attained due to antagonistic properties of the latter bac-
teria. Lactobacillus brevis 0944 strain contained in the
beetroot juice displayed antagonistic activity against many
bacteria (Escherichia coli, Pseudomonas aeruginosa,
P. Xuorescens, Bacillus subtilis and B. mycoides) and fungi
(Geotrichum candidum, Fusarium latenicum, Mucor hiemalis,
Aspergillus ochraceus, A. niger and Alternaria alternate)
[14]. The mutual antagonistic activity (i.e. against strains of
the same genus) of the strains contained in the starter
culture used for fermentation of the beetroot juice is weak.
It was conWrmed by the stabilization of the number of
Lactobacillus sp. cells (8.2–8.6 log units) observed in all
three groups of the experimental rats (Table 2). There were
no statistically signiWcant changes in counts of two groups
of bacteria (Bacteroides sp. and Enterococcus sp.). The
number of Clostridium sp. cells was increased (by 1.1–1.6
log units, P · 0.05) in the groups of rats, which consumed
1.5 and 3.0 mL of the fermented juice daily. The studies
provided evidence that supplementation of the diet of
experimental rats with lactic acid bacteria (6 £ 109 – 2.4 £

Table 2 Changes in cecal microbiota of rats fed with diVerent doses of fermented beetroot juice
Diet group Microorganisms, log CFU/ga,b (§SD)

Lactobacillus BiWdobacterium Clostridium Bacteroides Enterococcus Enterobacteriaceae Total number

sp. sp. sp. sp. sp. family of anaerobes

Casein diet (control) 8.2 (§0.63) 6.7 (§0.42) 8.0 (§0.35) 8.1 (§0.03) 7.3 (§0.43) 7.5 (§1.39) 9.90 (§0.90)
Casein diet+ 1.5 mL 8.6 (§0.95) 6.2 (§0.93) 9.1 (§0.42)* 8.3 (§0.61) 7.7 (§0.57) 6.7 (§0.48)* 11.0 (§0.40)*
fermented
beetroot juice
Casein diet+ 3.0 mL 8.2 (§0.53) 6.4 (§1.06) 9.6 (§0.96)* 8.6 (§0.50) 7.5 (§0.46) 6.1 (§0.42)* 10.9 (§0.19)*
fermented
beetroot juice
Casein diet+ 6.0 mL 8.3 (§0.64) 7.5 (§0.91)* 8.1 (§0.57) 8.0 (§1.17) 7.3 (§0.92) 5.4 (§0.04)* 10.8 (§0.13)*
fermented
beetroot juice
*Statistically signiWcant diVerence relative to the control group, P · 0.05
a
Average value n = 8
b
log10 CFU/g wet weight of faeces

123
156 Eur Food Res Technol (2009) 229:153–157

Table 3 Activity of selected enzymes of cecal microbiota of rats fed with diVerent doses of fermented beetroot juice
Diet group Enzyme activitya [U/g digesta] Enzyme activitya [U/g protein]

-Glucosidase
-Galactosidase
-Glucuronidase
-Glucosidase
-Galactosidase
-Glucuronidase

Casein diet (control) 0.81 2.72 2.49 98.2 334.1 304.3

Casein diet + 1.5 mL 0.64* 3.02 2.13 75.4* 361.5 261.3
fermented beetroot juice
Casein diet + 3.0 mL 0.46* 2.52 1.84* 53.6* 298.2* 224.9*
fermented beetroot juice
Casein diet + 6.0 mL 0.56* 2.77 2.03* 59.5* 301.7 218.9*
fermented beetroot juice
a
Average value n = 8
*Statistically signiWcant diVerence relative to the control group, P · 0.05

1010 CFU/dose) for 30 days stabilized the number of
Lactobacillus sp., Bacteroides sp., and Enterococcus sp.
cells. According to Garrido et al., who investigated modu-
lation of human intestinal microXora, the latter three genera
of bacteria and Bifodobacterium sp. were stabilized by
strain Lactobacillus johnsonii La1 [15]. Besides, the strain
La1 did not aVect the count of Clostridium sp. [15]. Biagi
et al., who studied in vivo (adult dogs) inXuence of
Lactobacillus animalis LA4, observed a decrease in the
count of Clostridium sp.; however, this variation was not
statistically signiWcant [16]. Researchers who examined the
impact of the consumption of yogurt (fermented by
Lactobacillus delbrueckii ssp. bulgaricus) on human
intestinal microbiota found no changes in test groups
probes: Clep 866 (Clostridium leptum, Ruminococcus,
Eubacterium) and Erec 482 (Clostridium, Ruminococcus,
Eubacterium, Butyrivibrio) [17]. The strains applied in this
study (Lactobacillus casei 0920 and Lactobacillus brevis
0944) stabilized the biWdobacteria. We observed the
decrease in a level of BiWdobacterium sp. from 6.7 log units
in the control diet group to 6.2 and 6.4 log units for animals
fed with 1.5 and 3.0 mL fermented beetroot juice daily, but
this diVerence was not statistically signiWcant relative to the
control diet group. We observed a signiWcant increase in the
number of cells of BiWdobacterium sp. to level 7.5 log units
only in the third group of animals, which consumed 6 mL
of our fermented product daily.
Consumption of fermented foods which contain live
cells of lactic acid bacteria can positively modulate intesti-
nal microXora and its metabolic activity, particularly
enzymes which participate in the process of carcinogenesis.
The group of cecal enzymes which are involved in conver-
sions of endogenous toxins and genotoxic compounds
comprises azoreductase, nitroreductase,
-glucuronidase,

-glucosidase,
-galactosidase, and 7--dehydroxylase
[18]. Bacteroides sp., Enterococcus sp., Clostridium sp.,
and Eubacterium sp. are eYcient producers of glycosidases
(
-glucosidase,
-glucuronidase,
-galactosidase) [19]. Our
study showed that not only the proWle of cecal microXora
but also the activity of selected microbial glycosidases were
changed in these animals which were fed with the basic
casein diet supplemented with the fermented beetroot juice
(Table 3). Activities of
-glucosidase and
-glucuronidase
were considerably reduced in all these rats. The activity of

Table 4 Concentrations of short chain fatty acids in the cecal digesta of rats fed with diVerent doses of fermented beetroot juice
Diet group Concentrationa [
mol/g] Concentrationa [
mol/100 g BW]

C2 C3 C4⬘ C4 C5⬘ C5 Total C2 C3 C4⬘ C4 C5⬘ C5 Total

Casein diet (control) 50.8 9.81 2.84 9.67 1.16 1.86 73.1 39.7 7.79 2.06 7.29 0.87 1.44 59.2
Casein diet + 1.5 mL 50.8 9.06 3.02 10.2 1.04 2.21* 73.4 44.5* 8.12 2.77 8.79* 0.96 1.96* 67.1*
fermented beetroot juice
Casein diet + 3.0 mL 48.7 10.1 3.12 10.6* 1.24 1.88 72.9 43.7* 8.89* 2.78 9.36* 1.10* 1.72* 67.6*
fermented beetroot juice
Casein diet + 6.0 mL 53.4 11.0* 3.37 11.3* 0.94 1.76 79.4* 50.5* 10.5* 3.42* 10.8* 1.01 1.78* 78.1*
fermented beetroot juice
SEM 0.43 0.17 0.05 0.15 0.03 0.04 0.70 0.99 0.27 0.12 0.32 0.02 0.04 1.73
BW body weight, C2 acetate, C3 propionate, C4⬘ isobutyrate, C4 butyrate, C5⬘ isovalerate, C5 valerate
*Statistically signiWcant diVerence relative to the control group, P · 0.05
a
Average value n = 8

123
Eur Food Res Technol (2009) 229:153–157

-glucosidase of control rats was 98.2 U/g protein. In ani-
mals fed with 1.5, 3.0, and 6.0 mL of fermented beetroot
juice daily, this activity was lower and reached 75.4, 53.6,
and 59.5 U/g protein, respectively (77, 55, and 60% of
activity of the control group). The activity of
-glucuroni-
dase was considerably reduced in intestines of these rats
which were administered with 3.0 and 6 mL of the fer-
mented beetroot juice daily (by 26 and 28%, respectively).
The comparison of changes in activity of the selected cecal
enzymes with concomitant changes in composition of intes-
tinal microXora of rats implies that these enzymes were
eYciently produced by bacteria of Enterobacteriaceae
family. Proliferation of the latter microorganisms was
strongly reduced in intestines of these rats which were fed
with the fermented beetroot juice and a drop in a number of
cells of Enterobacteriaceae strains was proportional to the
volume of the juice consumed per day.
The SCFA rank among important factors which aVect
the physiological state and “condition” of colonocytes of
intestinal mucosa. The role of butyric acid, which is a
source of energy for colonocytes, consists in stimulation of
diVerentiation of intestinal epithelium cells [20, 21]. Con-
centration of this acid was increased in intestines of all the
rats fed with the diet supplemented with the fermented
beetroot juice irrespective of its dose per day (1.5, 3.0, and
6.0 mL of juice) and the diVerence versus the control group
was statistically signiWcant (P · 0.05) (Table 4). Concen-
tration of butyric acid in the control group was 7.29
mol/
100 g BW while in groups fed with 1.5, 3.0, and 6.0 mL of the
juice per day it rose to 8.79
mol/100 g BW (20% increase),
9.36
mol/100 g BW (28% increase), and 10.80
mol/
100 g BW (48% increase), respectively. Butyrate acts as the
histone-deacetylase inhibitor and can modulate gene
expression, signal transduction, and protein degradation
pathways. In vitro studies using diVerent colorectal cell
lines demonstrated that butyrate induced cell cycle arrest,
diVerentiation, and apoptosis [22].
Conclusion
Regular intake of the fermented beetroot juice can contrib-
ute to stabilization of the intestinal microbial ecosystem.
157
Fermented beetroot juice can modify metabolic activity of
animals and thereby reduce the risk of diseases associated
with food intolerance.
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