African J. Biol. Sci.

, 7 (1): 1-14 (2011) ISSN 1687-4870

E.mail: aasdjournal@yahoo.com

Influence of various microbial starters of miso on lipid profile

and hstological and histoche mical changes of liver and kidney of mice

Abeer A. Abu Zaid1,2 , Amaal Mohammadein1,3 , Fawzia El-Salmy1

and Nahla S. El-Shenawy1,4*
1
Biology department, Faculty of Science, Taif Univ. (Qurwa), Taif, Saudi Arabia.
2
Food Technology Research Institute, Agriculture Research Center, Giza, Soy product Processing
Center, Egypt.
3
Zoology Department, Faculty of Science, Zagazig Univ., Egypt.
4
Zoology Department, Faculty of Science, Suez Canal Univ., Ismailia 41522, Egypt.
*Corresponding author: Zoology Department, Faculty of Science, Suez Canal University, Ismailia
41522, Egypt. *e-mail address: elshenawy_nahla@hotmail.com

ABSTRACT
The present study was undertaken to evaluate the effects of feeding the male mice
on different types of miso that prepared with mixture of microbial starters on body weight,
lipid profile, activities of lactate dehydrogenase and gamma glutamyl transferase.
Moreover, the effects of different types of miso on the liver and kidney at the histological
and histochemical levels were study. Miso (1) was prepared with Aspergillus oryzae and
Pleurotus ostreaus. Miso (2) was prepared with using A. oryzae and B. subtilis.
The results indicated that miso (2) increased most of the lipid profile parameters
significantly in serum of mice as compared to the control and to the miso (1) except the
levels of TL and TC of mice which were the same in both groups of miso. The activity of
leaking enzyme, LDH, was comparable between the animals of the two groups. The activity
of -GT was increased significantly in animals that feed on both types of miso as compared
to their level in the control animals. The results of histological analyses indicated that
absorption and transport of nutrients to liver and kidney were affected by the presence of
soybean products (miso) in the experimental diets. The large vacuoles observed in the
hepatocytes of mice fed miso (1) were filled with lipids. The histochemical study on the
kidney tissue of mice that were fed on miso (2) diet indicated accumulation of
carbohydrates. Health benefits from the use of natural products have evoked interest in
finding new and novel sources of such compounds for application in nutraceutical,
pharmaceutical and cosmetic industry.

Key words: Total cholesterol, Total lipid, Lactate dehydrogenase, Gamma-glutamyl

transferase, Histology, Histochemistry, mice, liver, kidney.

INTRODUCTION paste (Doenjang) has been traditionally

Most fermented soybean pastes are
salty and savory and some are spicy. They
are often used as condiments to flavor
foods such as stir-fries, stews, and soups.
Fermented soybean products may help to
prevent or attenuate the progression of
type 2 diabetes (Young Kwon et al.,
2010). Fermented soy foods, such as
paste, are important components of the
Korean diet in most of the world. Soy
manufactured from Koji, which is a
fermented rectangular block of crushed
cooked soybeans. The primary micro-
organisms involved in Koji fermentation
are Bacillus subtilis and molds such as
Rizopus, Mucor, and Aspergillus species
(Park and KO, 2005). Soy paste
fermented with various filamentous fungi
including Aspergillus awamori, A. oryzae,
A. sojae, Rhizopus azygosporus and R. sp.
2 Abeer A. Abu Zaid et al.
lead to the increased antioxidant
activities, total extractable phenolic
compounds and anthocyanins content
after fermentation (Lee et al., 2007).
Fermentation of plant ingredients
including soybean meal has been reported
to improve their nutritional value. In a
yellowtail diet, inclusion of soybean meal
fermented using either Aspergillus oryzae
or Eurotium repens improved the protein
and carbohydrate digestibility (Shimeno
et al., 1993). Naem (2010) studied the
effect of miso products that prepared by
single starter (Aspergillus oryzea) on
growth and blood serum constituents in
rats. The results revealed that the body
weight gain, total lipids, cholesterol, liver
function and kidney function were
significantly decreased compared with
control (basal diet). Miso caused an
increase in the amount of estrogen in the
female rats after feeding on miso for 2
months. Consumption of soy- products
reduced the low density lipoprotein
cholesterol (LDL-c) level; soy may serve
as a beneficial addition to a traditional
cholesterol- lowering diet by further
reducing serum cholesterol. Daily
consumption of 31 to 47 g of soy protein
has the potential to decrease the serum
and LDL cholesterol. Anderson et al.
(1995) reported a non-significant increase
in serum high density lipoprotein
cholesterol (HDL-c). Moreover, El-
Shenawy and Abu Zaid (2011) reported
that supplementation of diet with miso
that prepared with A. oryzea fungi in
both concentrations 10% and 20% to rat
were significantly decreased the levels of
total lipid, total cholestrol and LDL-c but
did not effect on some liver anzymes
activities.
However, preparation of miso with
two starters as A. oryzae and Bacillus
subtilis inhibited the proliferation of
human tumor cell lines culture with a
wide variation in LC 50 values (Abu Zaid
and E-Shenawy, 2010). On the other
hand, the influence of the starters in the
preparation of miso has been extended to
its effect on the endogenous antioxidant
of mice, after the animals fed on the miso
for 28 days (El-Shenawy et al., 2011).
Therefore the present study concern
on the effect of two types of miso that
prepared with different mixture of starters
on the body weight, lipid profile and
some liver enzymes biomarkers in mice.
Also, the histological and histochemical
alterations in the liver and kidney of mice
are discussed, particularly in comparison
to those of other control animals, in order
to determine the possible potential use of
these histopathological alternations as
indicator of miso effects on the cellular
level.
MATERIALS AND METHODS
1. Che micals and reagents
Commercial Kits of lactate
dehydrogenase (LDH) and total protein
were obtained from Human diagnostic
worldwide, German. Kits of total
cholesterol (TC), low-density lipoprotein
cholesterol (LDL-c), high-density
lipoprotein cholesterol (HDL-c), total
lipid (TL), triglycerides (TG) and gamma
glutamyl transferase (-GT) were
obtained from Biodiagnostic, B.D.,
Egypt. Other chemicals were procured
from Merck Ltd., SRL Pvt., Ltd.,
Mumbai, India.
2. Microorganis ms and Media
The microorganisms included in
this investigation are commonly used as
starters in the fermentation of many
traditional, oriental food products. The
mold Aspergillus oryzae, was used to
make traditional doenjang, a Korean
fermented soy paste (Kim et al., 2009),
and the bacterium Bacillus subtilis was
found among the microbial community in
doenjang (Yoo et al., 1999). Potato
dextrose agar was used for growth and
maintenance of all mold strains, and
nutrient agar medium was used for the
bacterium Bacillus subtilis.
 Influence of various microbial starters of miso on lipid profile
and hstological and histochemical changes of liver and kidney of mice
3. Preparation of miso
Miso was prepared in two major
steps. In the first step, microbial starters
were prepared as follows: wheat bran
seeded with Aspergillus oryzea fungi as
singly at approximately 1-2 x 106 cfu/g.
Then, it was spread at the surface of a
layer of 20 Kg of boiled and steamed
wheat at a ratio of 1% and incubated at 25
C for 3 days in order to encourage
growth and multiplication of microbial
starters. Secondly, the growing starter
was added to 40 Kg of boiled and
steamed soybean mixed thoroughly in
presence of 16% salt and moisture content
was controlled at approximately 12.5%.
The resulting paste represents the miso at
zero time. If the paste was stored in jars
for fermentation and aging, the resulting
miso represents 6 months or more (ISO,
2006).
Miso was also prepared with
different mixture of starters; Aspergillus
oryzae and Pleurotus ostreaus or A.
oryzae and B. subtilis (Abu Zaid et al.,
2011). The bacteria were routinely
cultured on nutrient agar and their stock
cultures were maintained at -80C in 20%
glycerol. For inoculums preparation, the
bacteria were grown in nutrient broth at
37C for 24 h. The cells were then
harvested, resuspended in sterile distilled
water and properly adjusted to obtain a
concentration of 104 cfu /mL. The
suspension was served as the inoculums
for soybean fermentation.
4. Che mical analyses of raw materials
and miso final products
These include measuring
moisture, crude protein, free amino acids,
fats and free fatty acids according to the
Official Method of Miso Analysis
(AOAC, 1995).
5. Animals and treatment protocol
Male mice (weighing 3035 g)
were purchased from King Fahed Medical
Research Center in Jeddah (Saudi
3
Arabia). The animals were maintained in
solid-bottom shoebox-type polycarbonate
cages with stainless steel wire-bar lids,
using a wooden dust-free litter as bedding
material. Animals were located in air-
conditioned room and were allowed free
access to pellet diet and tap water for a
week before starting the experiment. The
European Community Directive
(86/609/EEC) and National rules on
animal care have been followed. Animals
were randomly divided into 3 groups with
eight animals in each as follows:
1. Group 1 included mice fed on a
standard basal diet for 28 days
(Control group). This diet was
formulated according to Farag et
al. (2006) (Table 1) and water was
available ad libitum.
2. Group II included mice fed on
normal basal diet supplemented
with 10% miso (1); where the
10% of corn starch replace by
10% of miso. Composition of
experimental diet was shown in
table (1). Miso (1) in which
Aspergillus oryzae and Pleurotus
ostreaus were used as mixture of
starters.
3. Group III included rats fed on
normal basal diet supplemented
with 10% of miso; where the 10%
of corn starch replace by 10% of
miso (2) that prepared by using A.
oryzae and B. subtilis as mixture
of starters.
Animals in the three experimental groups
feed for 28 days.
6. Body weight and blood collection
Body weight of male mice was
recorded at zero, 2 and 4 weeks of the
experimental period (28 days). At the end
of each period, blood samples were
withdrawn from the animals under ether
anesthesia by puncturing the retro-orbital
venous plexus of the animals with a fine
sterilized glass capillary. Blood was
collected into non-heparinized tubes and
4 Abeer A. Abu Zaid et al.
left for 20 min at room temperature, then
centrifuged at 3000 rpm for 10 min using
Z 216 MK HERMLE refrigerated
centrifuge, to separate the sera. The sera
were kept in a deep freezer (~20 C) until
analyzed within three days.
7. Assessment of markers enzymes and
lipid profile
Biochemical analyses of serum
obtained from the collected blood were
conducted. Activities of serum LDH and
-GT and the levels of serum TL, TC,
LDL-c, HDL-c, TG and TP were
measured spectrophotometry (Jenway
6305 UV/VIS) using commercial kits.
8. Histological and histochemical
examination
At the end of each experiment, liver
and kidney specimens were removed
from mice. The preserved tissues were
processed by a routine histological
method (Drury and Wallingston, 1980);
dehydrated in alcohol series and
embedded in paraffin wax. For light
microscopy studies, paraffin sections of
liver and kidney of mice were cut into
section 5 m thicknesses by a rotary
microtome. The sections were stained
with Harris haematoxylin and eosin (H &
E) to observe the histopathological
changes in liver and kidney by using a
light microscope (Nikon E400) and
digital images were captured with a
Nikon 4500 Coolpix digital camera.
Detection and visual evaluation of hepatic
and kidney glycogen were carried out
histochemically by using Periodic acid
Schiffs (PAS) technique for the
demonstration of polysaccharides
(McManus, 1948). Then, mercury
bromophenol blue (Chapman, 1975) was
used for total protein identification.
9. Statistical methods
The results were represented as
means SE. The differences between
group mean values were evaluated by
Students t-test for unpaired observations.
The differences in the incidence of
pathological findings were evaluated by
Fishers exact probability test. Values
were considered to be significantly
different when P-value was less than 0.05.
RESULTS
The levels of the total carbohydrate,
total fat and total fibre were higher in
animals fed on miso (1) than those fed on
miso (2) but there was no significant
difference between the two groups (Table
2). Data of final body weights of male
mice subjected to different regime are
shown in table (3). Mice fed on miso (2)
supplemented diet had significantly
increased body weight gain relative to
those fed on miso (1) or on the basal diet
(control). There was significant difference
between body weight gain of mice fed on
miso (1) and miso (2).
Effect of miso that prepared with
different starters on lipid profile of mice
after 28 days of feeding was presented in
table (4). Miso (2) increased most of the
lipid profile parameters significantly in
serum of mice as compared to control and
miso (1) groups except the levels of TL
and TC were the same in both groups of
miso. However, the LDL-c decreased
significantly by 70.85% and 55.25% for
animals that fed on miso (1) and (2),
respectively (Table 4). There was no
significant difference in TP between all
the groups of mice (Table 5). The activity
of serum LDH (Table 5) was significantly
decreased to 35.42 6.48 and 19.23
3.73 U/l in animals that fed on miso (1)
and (2), respectively as compared with
mice fed on basal diet (58.89 6.23 U/l).
The activity of leaking enzyme, LDH,
was comparable between the animals of
the two groups. The activity of -GT was
increased significantly in animals that fed
on both types of miso as compared to its
level of control animals (Table 5).
 Influence of various microbial starters of miso on lipid profile
and hstological and histochemical changes of liver and kidney of mice 5

The overall morphological and Histological observations of the

histochemical conditions of the liver and
kidney are summarized in Figures (2 and
3). Histological investigation of sections
of kidney of mice fed on miso (1) and (2)
diet indicated no alternation and
somewhat similar to the tissue of control
kidney (Fig. 2 A, B and C). The cortical
renal tubules either proximal or distal of
kidney of mice fed on (miso 1 and 2)
exhibited normal architecture. Renal
corpuscles and medullary tubules of
kidney of mice fed on miso 1 and 2 are
exhibited somewhat normal like
appearance (Fig. 2 A, 2 and C). Deep
affinity for mercury bromophenol blue
reaction in the medullary tubules of
kidney of mice fed on miso (1) diet
reflected the high proteinic nature (Fig. 2
D, E and F). While PAS reaction was
strong in the kidney tissue of mice that
fed on miso (2) diet indicating
accumulation of carbohydrates (Fig. 2 G,
H and I).
normal liver mice revealed that the
hepatocytes have regularly or round
shaped and clearly stained nuclei with
nucleoli and the eosinophilic cytoplasm
with small vacuoles (Fig. 3A). The
hepatocytes of mice fed on miso (1) diet
exhibited dark stained nuclei and
cytoplasm with numerous large vacuoles
(Fig. 3B), while hepatocytes of mice fed
on miso (2) diet exhibited rapid mitotic
division represented by numerous
binucleated hepatocytes (Fig. 3C).
Histochemical studies revealed the
presence of moderate reaction of protein
and high infiltration of fat in the
hepatocytes of mice fed on miso (1)
compared with deep affinity of protein in
the binucleated hepatocytes in animals
fed on miso (2) and in control group (Fig.
3 D, E and F). PAS reaction was strong in
liver tissue of mice that fed on miso (1)
indicating high glycogen storage in their
hepatocytes as compared to hepatocytes
of mice fed on miso (2) and of control
group (Fig.
3G, H and
I).

Fig. 1: Effect of miso that prepared with mixture of starters on total protein in tissue of liver
and kidney of mice. Mice were fed on miso as supplemnted diet for 28 days. The results
were represented as mean S.E. (n=8).
6 Abeer A. Abu Zaid et al.

Fig. (2). A, Section of control kidney of mice (HE-stained) showing general structure of
kidney (100 X). CT, cortical tubules; RC, Renal corpuscles; MT, medullary tubules.
B and C, Horizontal section of kidney of mice fed on miso (1) and 2, respectively.
D, Section of control kidney of mice (mercury bromophenol blue-stained) for general
protein reaction.
E and F, Section of mice kidney fed on miso (1) and (2) diet, respectively, (bromophenol
blue-stained) showing no histochemical changes of protein reaction.
G, Section of control kidney of mice (PAS-stained) for general carbohydrate reactions.
H and I, Section of kidney of mice fed on miso (1) and miso (2) diet, respectively, (PAS-
stained) showing no histochemical changes of carbohydrate reaction.
 Influence of various microbial starters of miso on lipid profile
and hstological and histochemical changes of liver and kidney of mice 7

Fig. (3). A, Section of control liver of mice (HE-stained) showing general structure of liver (200
X).V, central lobular vein; Arrow; hepatocyte nucleus.
B, Section of liver mice fed on miso (1) showing the hepatocytes with darkly nucleus (arrows) and
round or elliptic vacuoles (arrow heads).
C, Section of liver mice fed on miso (2) showing the hepatocytes binucleated (arrows) and round or
elliptic vacuoles (arrow heads).
D, Section of control liver of mice (mercury bromophenol blue-stained) for general protein reaction.
E, Section of liver mice fed on miso (1) (mercury bromophenol blue-stained) showing the
hepatocytes with darkly nucleus (arrows) and numerous round or elliptic vacuoles (arrow heads).
F, Section of liver mice fed on miso (2) (mercury bromophenol blue-stained) showing deep reaction
of protein hepatocytes binucleated (arrows) and little round vacuoles (arrow heads).
G, Section of control liver mice (PAS-stained) for general carbohydrate reactions.
H, Section of the liver mice fed on miso (1) diet (PAS-stained) showing deep reactions of
carbohydrates, the hepatocytes with darkly nucleus (arrows) and numerous round or elliptic
vacuoles (arrow heads).
I, Section of the liver mice fed on miso (2) diet (PAS-stained) showing the hepatocytes binucleated
(arrows).
8 Abeer A. Abu Zaid et al.

Table 1: Diet composition (%) for the three experimental groups for mice
Ingredients (%) Basic diet Miso (1) Miso (2)
Casein 15 15 15
Corn oil 10 10 10
Cellulose 5 5 5
Miso - 10 10
Salt mixture 4 4 4
Vitamin mixture 1 1 1
Corn starch 65 55 55
Miso (1) and miso (2) in which A. oryzae and P. ostreaus or A. oryzae and B. subtilis
were used as mixture of starters, respectively.
Table 2: Chemical Composition of different types of miso (g/100 g dry weight,%)

Total
Soy Total Total Total Total fi ber Total
Carbohydrate
paste nitrogen Fat Moisture Ash
(code)

Miso (1) 15.32 2.1 19.31 3.1 5.33 0.5 38.42 2.1 5.81 0.3
0.49 0.1

Miso (2) 13.0 1.6 13.82 1.4a 3.83 0.2a 41.21 1.8a 2.52 0.1a 0.44 0.1

P- Value -- 0.05 0.02 0.01 0.001 --

All data are expressed as mean SE (n=3). a P < 0.05, compared with group of miso (1).

Table 3: Amino acids content of miso final products after fermentation

Amino aci d (% ) Miso (1) Miso (2)

Asparatic 3.41 2.99

Therionine 1.23 1.04
Serine 1.56 1.25
Gl utamic 5.29 5.55
Proline 1.67 1.40
Gl ysine 1.28 1.01
Alani ne 1.33 1.18
Cystein 1.2 1.00
Valine 1.60 1.36
Methi onine 1.0 1.00
Isoleucine 1.38 1.32
Leucine 2.21 2.36
Tyrosine 1.04 0.94
phynelal anine 1.65 1.57
Histi dine 0.60 0.48
Lysine 0.82 0.67
Arginine 1.51 1.40
Total A.A 28.6 26.52
 Influence of various microbial starters of miso on lipid profile
and hstological and histochemical changes of liver and kidney of mice 9

Table 4: Effect of miso that prepared with different starters on body weight gain of mice.

Groups Initial weight (g) Weight (g) Final weight (g)

after 14 days after 28 days
Control 17.51 0.66 17.8 0.74 18.35 0.74
Miso (1) 22.65 0.51 22.48 0.98a 23.10 1.50 a
Miso (2) 21.70 1.64 22.35 1.51a 28.60 0.90 a,b
a
All data are expressed as mean SE of 8 mice. P < 0.05, compared with control
b
group. P < 0.05, compared with group of miso (1).

Table 5: Effect of miso that prepared with different starters on lipid profile of mice after 28
days of feeding.
Groups TL TC LDL-c HDL-c TG
(g/dl) (mmol/l) (mmol/l) (mmol/l) (mmol/l)
Control 0.64 9.01 46.73 6.62 0.38 0.01
0.01 0.71 0.94 0.64
Miso (1) 0.65 11.51 13.62 23.89 0.49 0.03
a a a
0.02 0.17 0.45 0.37
Miso (2) 0.72 12.43 20.91 32.73 0.82 0.06
b a a,b a,b a,b
0.02 0.27 0.32 1.34
All data are expressed as mean SE of 6-8 mice. aP < 0.05, compared with control group.
b
P < 0.05, compared with group of miso (1).
TL, total lipid; TC, total cholesterol; LDL-c, low-density lipoprotein cholesterol; HDL-c,
high-density lipoprotein cholesterol; TG, triglycerides.

Table 6: Effect of miso on activities of some biomarker for liver enzymes and level of total
protein in the serum of mice after 28 days of feeding.
Groups LDH (U/l) -GT (U/l) TP (g/dl)
Control 58.89 6.23 16.51 2.05 6.68 0.36
Miso (1) 35.42 6.48 a 32.27 4.13 a 7.87 0.57
Miso (2) 19.23 3.73 a,b 29.24 1.88 a 6.14 0.29

All data are expressed as mean SE of 6-8 mice. a P < 0.05, compared with control group.
b
P < 0.05, compared with group of miso (1).
LDH; lactic dehydrogenase, -GT; gamma glutamyl transferase and TP; total protein.

DISCUSSION of total nitrogen, carbohydrate, fat, and

The first aim of the present study
concerned on the levels of moisture,
crude protein, free amino acids, fats and
free fatty acids in different types of miso
that were prepared with different types of
mixture starters. The results proved that
miso (1) prepared with Aspergillus oryzae
and Pleurotus ostreaus has higher content
fiber than miso (2) that was prepared with
A. oryzae and Bacillus subtilis. However,
the percentage of total amino acid content
of both miso was approximately the
same.
The second aim concerned on the
evaluation the effect of different types of
miso on lipid profile, histological and
10 Abeer A. Abu Zaid et al.
histochemical changes in liver and kidney
tissues of mice. There was no data
concerning the influence of miso that
prepared with mixture of starters on
cellular structure for this reason it is very
difficult to compare our results with those
reported by other authors.
Fungi are of excellent value
nutritionally, and of great importance to
vegetarians. Edible mushrooms are good
source of protein, have low- fat content
and free of cholesterol. Edible
filamentous fungi producing hydrolytic
enzymes present an added advantage for
their use in food and feed (Ghorai et al.,
2009). Pleurotus ostreaus (Oyster
mushroom) has unique flavor and
aromatic properties; considered to be rich
in protein, fiber, carbohydrates, vitamins
and minerals. Aspergillus oryzae is a
filamentous fungus used in various
Japanese traditional fermented. A. oryzae
is also used in the production of various
industrial enzymes such as amylases and
proteases because it has good potential for
the high production of such enzymes
(Matsushita-Morita et al., 2010). Bacills
natto and B. subtilis produced hydrolytic
enzymes, such as proteases, mannanases
and glucanases, according to the culture
medium used.
The role of amino acids as the
major structural and functional
components of the body is well known,
and much research on the function of
essential amino acids, non-essential
amino acids, and other various peptides
has been conducted the amino acid
composition in relation to protein
nutritional quality of food groups (Juan et
al., 2004). Recently, the expectation for
the possible utilization of amino acids as
a dietary supplement for preventing or
treating chronic and/or metabolic diseases
has been brought up in Korea. In the past,
amino acid supplement was used only for
the professional athletes restrictedly to
improve their performance capacity but
now, it was present in fermented soy food
(miso).
In the present study, increasing
the body weight significantly was notice
after 28 days of feeding time with
supplemented diet with miso (2). Soya
protein breakdown by fermentation could
be an important factor for the
improvement in weight of mice.
Miso was prepared with microbial
starter as Aspergillus oryzea fungi and
stored for 6 months has the highest
amount of total isoflavone (daidzein and
genestein), amino acids and fatty acid as
compared to other miso that stored for
zero time or for 5 years (El-Shenawy and
Abu Zaid, 2011). Moreover, this type of
miso improved the liver functions and it
enhanced the antioxidant state of rat. All
the fermented soybeans products with
different mixture of starters contained
higher isoflavones compounds than
unfermented cooked soybeans. Moreover,
soybean fermented with B. subtilis
showed the highest amount of isoflavones
(Abu Zaid and El-Shenawy, 2010).
In the present study, serum TL
level of mice did not change after 28 days
of feeding the animals with miso (1) or
(2), while TC and HDL-c levels were
increased and LDL-c was decreased.
However, in the previous study, rats fed
on supplemented diet with 10% and 20%
of miso that prepared with single starter
(A. Oryzea) have significantly decreased
in the level of TL, TC and LDL-c (El-
Shenawy and Abu Zaid, 2011). Similar
decrease of LDL-c was described
previously in hamster fed with soybean
(Anosike et al., 2008). McVeigh et al.
(2006) showed that people whose diet
high in soybean protein has significant
reduction in serum TC and LDL-c. It is
clear that effect of miso that prepared
with mixture of starters were differed
from the effect of miso that prepared with
one starter only. Zhan and Ho (2005)
reported that soy protein containing
isoflavones can significantly reduced
 Influence of various microbial starters of miso on lipid profile
and hstological and histochemical changes of liver and kidney of mice
serum level of LDL-c. This decrease
could be due to the fatty acids content of
soybean that increase the activity of
receptors of low density lipoproteins on
adipose cells (Nelson et al., 2005).
TL levels did not change in the
present study during feeding mice with
miso (1) or (2). This effect of miso on TL
levels could be due to reduction in levels
of LDL-c and increasing the level of
HDL-c at the same time. This observation
is in agreement with Murray et al. (2003)
who reported that the total amounts of
lipids in blood usually remain at constant
percentage. Experimental research
consistently showed that soy foods cause
a decrease in bad LDL-c, and an increase
in good HDL-c (Jenkins, 2002). The miso
that was prepared with A. oryzae and
Bacillus Subtilis has more effect on
suppressing the oxidative stress and
enhancement of endogenous antioxidant
of hepatocytes and renal tissue of mice
(El-Shenawy et al., 2011). In the present
study the level of HDL-c in mice fee on
miso (2) diet was more than that in
animals that fed on miso (1).
The overall morphological
conditions of the examined kidney of
mice fed on miso 1 and 2 diets indicated
no apparent histological changes in all
sections of the kidney. Similar
observation has been reported by
Yamamoto et al. (2010) who found that
there was no histological changes in the
kidney of trout fed on soybean meal diet.
In the present study mild
abnormalities were found in the liver of
some mice feed on miso 1 and 2 diets.
Large vacuoles filled with lipids were
observed in the hepatocytes of mice fed
on miso (1). In addition, PAS-positive
granules (glycogen storage) were
similarly observed in the cytoplasm of
hepatocytes of mice fed on miso 1 and 2
diets, suggesting that the vacuoles, which
decreased in the cytoplasm of hepatocytes
in mice fed the fermented soybean meal
diet, mainly store lipids.
11
The effect of fermentation of
plant ingredients including soybean meal
on the improvement of their nutritive
values could be attributed to the decrease
in the amounts of antinutritional factors
present in the ingredients. Refstie et al.
(2005) reported that oligosaccharide
content of soybean meal decreased by
fermentation with A. oryzae or E. repens
and with L. brevis, respectively, and
digestibility of certain nutrients increased.
Fermentation of soybean meal by Bacillus
subtilis for 48 h at 37 C decreased the
molecular weights of soya proteins (Feng
et al., 2007). In rats, soya proteins
undigested by microbial proteases has a
strong hypocholesterolemic effect, but
further digestion of the proteins by
various proteases diminished this effect
(Sugano et al., 1990). In mammals, one of
the hypocholesterolemic effects by
dietary soybean has been attributed to the
increased excretion of bile acids, which
are synthesized from cholesterol in the
liver (Potter, 1995). From these findings,
it is likely that soya proteins in the present
study had been digested, the extent of
which was not identified, during the
fermentation by compound bacteria
including Bacillus sp. and had lost the
property of binding bile acids.
The hepatocytes of soybean meal
and soybean protein concentrate-based
diet fed rainbow trout showed anomalies.
In rainbow trout and juvenile South
American pacu, the average hepatocyte
nuclear volumes was significantly
differed among the feeding groups. The
results of histological analyses indicated
that absorption and transport of nutrients
to liver and pancreas were affected by the
presence of soybean products in
experimental diets (Ostaszewska et al.,
2005). The present results agreed with
Dajanta et al. (2009) who suggested a
promising use of Bacillus starter cultures
in improving isoflavone compounds
which would benefit for novel functional
food development. B. subtilis fermented
12 Abeer A. Abu Zaid et al.
soybeans appear to be a better source of
bioavailable soy isoflavones (El-Shenawy
et al., 2011) as it has been reported that
aglycone isoflavones are absorbed faster
and in higher amounts than their
glucosides in humans (Kano et al., 2006).
In conclusion, the present study
gives an insight into the miso (2)
improved the body weight, level of LDL-
c and the activities of LDH and -GT than
miso (1) in male mice. There was no
histological and biochemical changes in
the kidney of mice that fed on two types
of miso.
Acknowledge ments
This study was financially
supported by funding source from the
Scientific Research Unit of Taif
University, Taif, KSA. This study was a
part of the grant No 1-431-644.
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41
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