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African Journal of Food Science Vol. 5(14), pp.

775-779, 30 November, 2011

Available online
ISSN 1996-0794 ©2011 Academic Journals
DOI: 10.5897/AJFS11.128

Full Length Research Paper

The effect of black soybean tempe and it’s ethanol extract

on lymphocyte proliferation and IgA secretion in
Salmonella typhimurium induced rat
1 2 2 3
Nurrahman , Mary Astuti , Suparmo and Marsetyawan, N. H. E. Soesatyo
Semarang Muhammadiyah University, Semarang, Indonesia.
Faculty of Agricultural Technology, Gadjah Mada University, Yogyakarta, Indonesia.
Faculty of Medicine, Gadjah Mada University, Yogyakarta, Indonesia.
Accepted 1 November, 2011

Children patient suffering from malnutrition and chronic diarrhea, who had been treated with tempe formula
improved their health status, recovered from the diarrhea in a relatively shorter period, and gained weight. It is
perceived that the quick recovery was due to improvement of the body’s immune system. The aim of the research
was to observe the effect of black soybean tempe and it’s ethanol extract on T and B cells proliferation and level of
secretory IgA (sIgA) in rat induced with Salmonella typhimurium. A total of 48 rats were divided into four
groups, 12 rats each assigned to standard diet, diet plus black soybean tempe flour, diet plus tempe ethanol extract,
and diet plus mixture from tempe ethanol extract and flour of black soybean tempe. Body weights were recorded
during 35 days of diet treatment, and index stimulation on T and B cells as well as level of secretory IgA were
measured after S. typhimurium induction. Result indicated that diet with tempe, diet with ethanol extract and
combination of both increased body weight better than that of control. Treatments with tempe and mixture of tempe
with its ethanol extract were significantly different from the control in the proliferation of T cell; however, the
treatment with tempe ethanol extract did not. The three treatments did not improved proliferation of B cell. Diet
with tempe alone significantly increased secretory IgA in the intestinal tract fluid of rats, but the other treatments
did not show significant difference with the control (without Salmonella induction).

Key words: Black soybean tempe, immune response, lymphocyte, secretory IgA.

Tempe, an Indonesian body capacity to defence

traditional food, made from against free radical attact
soybeans through (Astuti, 1997).
fermentation with Rhizopus Due to the availability of
fungi, is respected as a high soybeans in the world
quality protein food derived market and the imbalance of
from plant origin, excellent high consumption and low
digestibility, low in production of soybeans in
antinutrition factors, such as the country, tempe in
phytic acid, Indonesian markets are
oligosaccharides, and good mostly processed from
source of complex of imported yellow soybeans.
vitamin B (Suparmo and The black soybeans, native
Markakis, 1987). The variety that re-gained its
traditional food is regarded popularity locally, were
as health food due to its proved to have more
content of several beneficial effect
antioxidants, such as
nutritionally. Xu and Chang
genestein, daidzein and
(2007) indicated that black
isoflavones. Tempe also
soybeans contained phenolic
modulates internal
antioxidant super oxide substances, tannins,
dismutase (SOD), which isoflavones higher than the
improve yellow variety and they also
had higher antioxidant
activity, as well. Astadi et al.
(2009) found that
*Corresponding author. E-mail: anthocyanin in the black soy hull inhibited the oxidation
Tel: 6202476482951/ of low density lipoprotein
08122502964. Fax: (LDL) in an in vitro test.
776 Afr. J. Food Sci.

In some Indonesian hospitals, tempe porridge is used by (pure culture of R. stolonifer DUCC204) and way of hull separation (10%
nutritionist experts as one of diarrhea therapy for children. of the hull was remixed with the cotiledone). Black soybean were cleaned
to remove dirt and foreign objects, washed with water, soaked for 30 min,
Research conducted by Hermana et al. (1996) indicated that and then boiled with water for 30 min. Boiled soybeans were dehulled by
children suffering from malnutrition and chronic diarrhea that hand and 10% of the hull was put back with the cotiledone, soaked for 36
were given tempe formula had improved their nutritional status, h, thoroughly drained, and then steamed for 1 h. The steamed dehulled
gained weight, and healed from the diarrhea in a relatively soybeans were allowed to cool for 30 min prior to inoculation with R.
shorter period. This is likely related to the recovery of the body stolonifer DUCC204 starter. The ratio of inoculum to soybeans was 2 g of
starter for every kg of soybeans. The inoculated soybeans were packed in
immune system (immune) both systemically and locally in the
plastic bags (with tiny holes in it) and incubated at room temperature (25
digestive tract. Immune responses against antigens that enter to 27°C) for 36 h. The resulted tempe was then dried at temperature of 40
the body are influenced by genetic factors, environment, and to 45°C for 24 h and ground to tempe powder (60 mesh).
foods. Several researches had been conducted to study the
effects of food on the immune system (Nurrahman et al., 1999;
Tejasari, 2007; Zakaria-Rungkat et al., 2003). It is related to the
active components of both nutritional and non-nutritional Antioxidant extraction of tempe powder with ethanol
substances in foods that support formation of the body's
defense system. Antioxidant extractions were conducted upon black soybean tempe
powders according to the method of Xu and Chang (2007). Tempe
powders (100 g) were put in erlenmeyer flasks, mixed with 200 ml of
ethanol 70% and let to stay for 24 h. The extracts and the remaining mass
Xu and Chang (2007) indicated that phenolics, thanins and were separated by way of filtration. The mass was mixed again with 100
isoflavones, and antioxidant activity in black soybeans were ml of 70% ethanol and then refiltrated. The two filtrates were combined
higher than those of the yellow ones one. The superiority of the and the solvent was evaporated to obtained dry extract.
black soybeans and numerous benefits of tempe fermentation,
demonstrated by researches in the past, have triggered the
curiousity in studying the benefits, especially in nutritional and Care of rats
health aspects, of consuming tempe made of black soybeans.
This research was aimed to study the effect of black soybean Ethical clearance (approval) was obtained from Ethical Committee of
tempe and its antioxidant (ethanol) extract on T and B cells Research in Medical Health of Faculty of Medicine Gadjah Mada
University for the study for lymphocyte and IgA study. A total of 48 male
proliferation and content of secretory IgA in rats induced with Wistar rats, age 6 to 8 weeks, obtained from Gadjah Mada University
S. typhimurium. Animal Laboratory were divided into four groups, 12 rats each. Each
group was assigned to different treatment diet (Table 1). The rats were
placed in individual cages at room temperature of 25 to 27°C and
aclimated on standard diet for 5 days prior to feeding treatment. The
MATERIALS AND METHODS animals were then kept on treatment diets for 28 days. Both diet and water
were provided ad libitum. Body weight during feeding treatment was
The materials used in this research were black soybean tempe (Mallika, monitored.
breed from native variety), antioxidant (ethanolic) extract of the tempe,
culture of S. typhimurium FNCC0135, rats (wistar, male, 6 to 8 weeks old
of 44.2 to 54.9 g in weight), and some reagents such as Roswell Park Preparation of Salmonella typhimurium culture
Memorial Institute (RPMI) 1640 (RPMI-1640, Sigma, USA), IgA Rat Kit
(Immunology Consultants Laboratory, Inc, USA), Phytohemagglutinin The preparation of Salmonella culture was conducted by inoculating 300
(PHA) (PHA, Sigma, USA), Lipopolysaccharides (LPS) (LPS, Sigma, ml of nutrient broth with 3 ml of the bacteria suspension. After incubation
USA), Vitamin mix AIN 93 and Mineral mix AIN 93. at 37°C for 20 to 24 h the optical density of the suspension was measured
at 600 nm. At absorbance of 0.5 the population is 10 cfu/ml (by standard
curve previously prepared). The suspension was centrifuge at 3500 rpm for
15 min, the pellet was resuspension in 30 ml NaCl 0.85%, the suspension
Tempe starter preparation 9
concentration became 10 cfu/ml.
Rice and water in a ratio 1:1 by weight were put in an erlenmeyer flask,
and then sterilized at 121°C for 15 min. The rice was allowed to cool at
room temperature and then inoculated with 1 ml suspension of Rhizopus Preparation of intestinal fluid before induction with Salmonella
stolonifer DUCC204 (source of Rhizopus Diponegoro University Culture typhimurium
Collection, Indonesia) having 10 cell. The inoculated rice was spread in
containers and covered with aluminium foil. Tiny holes were made on the A total of 24 rats (6 from each group) were sacrifized and their small
aluminium cover to provide micro aeration. The mixture was incubated at intestine were separated. Extraction of intestinal fluid was conducted by
room temperature for 3 days, dried, and then pulverized to fine tempe injecting 5 ml phosphate buffer saline (PBS) through the tube of the
starter powder. intestine, after which the fluid that passed the intestine were collected.

Processing of black soybean tempe Preparation of lymphocyte and intestinal fluid after induction with
Salmonella typhimurium
The tempe preparation adopted in this research was the regular way of
tempe making in Indonesia with modification in the inoculum The remaining 24 rats were inoculated with 1 ml of S. typhimurium
Nurrahman et al. 777

Table 1. Composition of diet (g/ kg).

Composition Diet of standard Diet of tempe Diet of tempe Diet of tempe + tempe
ethanol extract ethanol extract
Casein 140 - 140 70
Corn starch 620.7 561.66 620.7 591.18
Tempe flour - 278.5 - 139,25
Antioksidant extract - - 28 14
Soybean oil 42.29 - 42.29 21.145
Sucrose 100 100 100 100
CMC 50 35.4 50 42.7
Vitamin mix AIN 93 10 10 10 10
Mineral mix AIN 93 35 35 35 35
L-cystin 1.8 1.8 1.8 1.8
Kholin bitatrat 2.5 2.5 2.5 2.5
incubated under 95% humidity, Growth of rats during
5% CO2 atmosphere, and experiment
temperature of 37°C. After 72 h of
9 incubation, 10 µl of MTT solution Absorbance of mitogen
suspension containing 10 cfu/ml
of 5 mg/ml were added into each
Body weight of rats at the
per oral. After 1 weeks on treatment beginning of the trial was
well using micro pippette. The IS =
corresponding treatment diets, the were between 44.2 to 54.9 g.
incubation was continued for Absorbance of
rats were sacrifized to obtain their Changes in body weight
another 4 h. Living cells reacted control
small intestines, Peyer’s patches,
and spleens. The small intestine
with MTT to form a purple color. during maintenance with diet
Reaction with MTT was stopped treatment are presented in
were treated with PBS to obtain
by adding 100 µl of stopper Figure 1. All of rats
the fluid for the determination of
reagents containing 10% sodium Measurement of
IgA after induction. The spleens experienced growth during
dodecyl sulfate (SDS) solution in immunoglobolin (IgA) with
and Peyer’s patches were
0.01 N hydrochloric acid. The the experiment (p<0.05). It
individually put in Petri dish indirect ELISA method
absorbances were read using showed that rats under
containing RPMI1640 media for treatment diets
microplate reader on 550 nm
the observation of lymphocyte
wavelength. Index of stimulation The measurement of
proliferation and IgA with indirect
indicating the lymphocyte immunoglobulin were conducted
ELISA method, respectively.
proliferation was calculated as according to IgA rat kit
follows: Bioscience manufacturer’s
procedure. Samples were
Isolation of lymphocyte from intestinal fluid and isolated
spleen and Peyer’s patch lymphocyte from Peyer’s patch.
Sample (5 ml) from intestinal
The spleen and Peyer’s patch fluid was centrifuged at 3000 rpm
were washed with RPMI media for 5 min and the filtrate was
three times, extracted by injecting ready for IgA analysis. Sample
10 ml of RPMI media into the from isolated lymphocyte from
spleen by a syringe. Suspension Peyer’s patch was adjusted to
came out from the spleen or obtain 3 × 10 cells/ml. An
Peyer’s patches consisting the amount of 200 µl of sample was
media and the extracted put in 96-well microplate and
lymphocyte. The suspensions incubated for 72 h. The
were collected into 15 ml lymphocyte suspension from
centifuge tubes, centrifuged at Peyer’s patch was ready to be
3000 rpm for 5 min, the filtrates used as sample for IgA analysis.
were removed, the pellets were
resuspensed in 10 ml of RPMI, Standards (rat IgA calibrators)
recentrifuged in the same manner, and samples (from fluid intestine
filtrates were removed, and the and lymphocite culture) were
remaining pellets were resuspesed taken, 100 µl each, and put into
again using complete media different labelled wells, which
consisting RPMI and fetal bovine already contain antigen. The plate
serum (FBS) 10%. were incubated at room
temperature 60 min, content of
wells were discarded, washed
with washing solution three times.
Measurement of lymphocyte An amount of 100 µl of antibody
proliferation enzyme conjugate were added
into each wells and incubated at
The number of lymphocyte room temperature for 30 min in a
suspension from spleen were dark room. Wells were washed
counted using haemocytometer again as previously done.
and then diluted to the Tetramethylbenzidine substrate
6 (TMB, Immunology Consultants
concentration of 1.5 10 cells/ml.
Laboratory, Inc, USA), 100 µl,
Lymphocyte suspension from
were added into each of wells,
control and the three feeding
incubated at room temperature in
treatments were filled into the 96
a dark room for 10 min, and then
wells microplate, 200 µl each.
mixed with 100 µl stop solution
Lymphocyte suspension taken
from a single rat was assigned to (0.3 M H2SO4). The micro plates
9 wells, so a total of 9 × 24 wells were read using microplate reader
were used (3 plates). The 9 wells (Bio_Rad, Model 680 XR, USA)
of lymphocyte suspension from at a wavelength of 450 nm.
single rat were divided into three
treatments, 3 wells were added
with 10 µl containing 5 µg of
PHA solution, another 3 were
added with 10 µl solution RESULTS AND
containing 1 µg of LPS, and the DISCUSSION
last 3 were added with 10 µl of
complete media. Lymphocyte
suspensions in the plates were
778 Afr. J. Food Sci.

Figure 1. Graphic effect of diet and long of maintenance against growth of body weight of rats.
significantly gained more genestein, an isoflavone
weight than those on the contained in soy and soy
standard diet (p < 0.05), products (tempe), was able
while there were no to bind to estrogen receptors,
significant difference among while Zao et al. (2005),
treatments. Rats fed on based on in vivo experiment,
tempe and ethanol extracts mentioned that components
of tempe formulas in soy products may poses
experienced higher growth the ability to interact with
than the standard one. This receptors on the surface of
shows that the nutrients the T cell to increase its
contained in tempe played a proliferation. Ramprasath et
role in the growth of rats. al. (2005) and Rimbach et al.
The findings were consistent (2008), reported that
with the result of Hermana et nutrition nutrients in feed
al. (1996) that consuming improved wellbeing, resulted
diet of tempe formula affect in higher weight gain and
weight gain faster in children may improve immune
suffering from malnutrition. system of the animal.

The B cell proliferation

indexes showed that rats
Proliferation of T and B which consumed feed with
cells black soybean tempe flour as
a substitution for casein,
The T cell stimulation tempe ethanol extract and
indexes of rats spleen after their combination with
35 days of diet treatments is ethanol extracts produced
presented in Table 2. T cell stimulation higher than
stimulation index of rats standard diet, however, there
spleen after treatment ranged were not statistically
from 0.888 to 1.301. Rats different (p > 0.05).
that consumed tempe and
tempe combined with it’s
ethanol extract had higher
indexes than those of Content of Secretory IgA
standard (p <0.05), however,
treatment with ethanol IgA content of intestinal
extract alone did not. This fluid in rats treated with
may be due to insufficient tempe diet and its
amount of extracted combination with ethanol
substances to create the extracts looked higher than
difference. It also indicates those of standard. This
that substance present in occured in rats both before
tempe other than those and after induction with S.
extracted with ethanol may typhimurium, which has the
poses the activity of same pattern (Table 2).
However, statistical analysis
increasing the index. Dixon
with ANOVA methods
and Ferreira (2002) found
indicated that diet
that, in in vitro experiment, treatment’s
Nurrahman et al. 779

Table 2. Effect of diet treatment on rats against index of stimulation and secretory IgA.

Parameters Type of diet

Standard Tempe Tempe extract Tempe + extract
Index of stimulation from T cells 0.936 ± 0.030 1.089 ± 0.106 1.034 ± 0.057 1.047 ± 0.082
Index of stimulation from B cells 0.986 ± 0.093 1.416 ± 0.349 1.161 ± 0.247 1.274 ± 0.368
Secretory IgA of intestine fluid (ng/ml)a 48.600 ± 26.445 73.900 ± 18.499 74.8 00 ± 26.530 55.133 ± 25.533
Secretory IgA of intestine fluid (ng/ml)b 50.533 ± 23.322 81.533 ± 17.319 76.367 ± 21.520 56.800 ± 26.977
Secretory IgA of lymphocytes from 120.467 ± 43.602 130.667 ± 41.221 94.4667 ± 49.270 70.017 ± 30.216
peyer’s patches (ng/ml)
before induced by Salmonella typhimurium, bafter induced by Salmonella typhimurium.
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