Effect of Fermented Soybean, "Natto" on the Production and Qualities of Chicken Meat

Abstract

Natto is a Japanese traditional soybean product fermented by Bacillus natto. The effect of natto supplement on the production and qualities of chicken meat was studied since the effective use of various waste foods is required in Japan. Dried natto prepared by heating at 60°C was added to a basic diet at an amount of below 2%. The supplementation of dried natto did not influence the weights of the carcass, breast and thigh meat, fillet or abdominal fat. Growth of the thighbone such as the length, thickness of cortex bone, and Ca/P ratio in bone ash were not altered by the addition of natto. However, the pH of male meat decreased following the supplementation of dried natto from days 28 to 80. The water-soluble protein content in male thigh meat increased in the group fed 2% natto from days 28 to 80. Free peptides increased in male thigh meat by feeding 2% natto from days 0 to 80. The supplementation of natto increased free glutamic acid in thigh meat regardless of sex. Moreover, the supplementation of natto specifically decreased meat cholesterol in female chickens though the effect was not shown in male chickens.

Full text

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INTRODUCTION
Since ancient times fermented soybean products were
made traditionally in Far East Asia including “Tempe” in
Indonesia, “Doubanjiang” in China, “Duenjang” and
“Chung-Kook-Jang” in Korea, and “Miso” and “Tofu-yo”
in Japan, (Asututi et al., 2000; Hong et al., 2004; Mine et al.,
2005). Natto is a Japanese cheese-like product also made
from soybeans by fermentation with Bacillus subtilis var.
natto (Bacillus natto), which is a gram-positive spore-
forming bacterium (Ashiuchi et al., 1998).
Currently more than 80% of feedstuffs for poultry are
imported in Japan (Ishibashi and Yonemochi, 2002). In
addition, large quantities soybeans are imported and the
self-sufficiency ratio is only 8% in Japan. Therefore, the
effective use of waste fermented soybean products with no
commercial value would be highly beneficial as feed (Cho
et al., 2007; Kim et al., 2007). Moreover, there are
advantages of the use of natto as feedstuff because the
biological value of soybeans is relatively higher than other
vegetable proteins and abundant nutrients remain after
fermentation such as isoflavones, saponins, lecithin, and
various vitamins.
In addition to residual nutrients, there are various
functional ingredients such as nattokinase, polyglutamic
acid, and dipicolinic acid in natto after fermentation. For
example, nattokinase is known as a fibrinolytic enzyme,
which cleaves directly cross-linked fibrin in vitro (Fujita et
al., 1993; Suzuki et al., 2003; Yamashita et al., 2003), and
polyglutamic acid is capable of calcium solubilization.
(Tanimoto et al., 2001) It is reported that the anti-
Helicobacter pylori activity of natto is derived from
dipicolinic acid (Sumi et al., 2006). Moreover, Bacillus
natto as a probiotic could be regarded as a functional
ingredient in natto. If natto could be used as poultry
feedstuffs, converting surplus natto into feedstuffs could
reduce feedstuff imports and productivity reuse food waste
material.
In this study, natto supplement was fed to “Tsukuba-
jidori”, which is a high-quality meat-type chicken. “Jidori”
Asian-Aust. J. Anim. Sci.
Vol. 21, No. 12 : 1766 - 1772
December 2008
www.ajas.info
Effect of Fermented Soybean, “Natto” on the Production and
Qualities of Chicken Meat*
K. Fujiwara, Y. Miyaguchi1, **, X. H. Feng1, A. Toyoda1, Y. Nakamura1
M. Yamazaki2, K. Nakashima2 and H. Abe2
Laboratory of Poultry, Ibaraki Prefectural Livestock Research Center, Ibaraki, 311-3157, Japan
ABSTRACT : Natto is a Japanese traditional soybean product fermented by Bacillus natto. The effect of natto supplement on the
p
roduction and qualities of chicken meat was studied since the effective use of various waste foods is required in Japan. Dried natto
prepared by heating at 60°C was added to a basic diet at an amount of below 2%. The supplementation of dried natto did not influence
the weights of the carcass, breast and thigh meat, fillet or abdominal fat. Growth of the thighbone such as the length, thickness of cortex
bone, and Ca/P ratio in bone ash were not altered by the addition of natto. However, the pH of male meat decreased following the
supplementation of dried natto from days 28 to 80. The water-soluble protein content in male thigh meat increased in the group fed 2%
natto from days 28 to 80. Free peptides increased in male thigh meat by feeding 2% natto from days 0 to 80. The supplementation of
natto increased free glutamic acid in thigh meat regardless of sex. Moreover, the supplementation of natto specifically decreased meat
cholesterol in female chickens though the effect was not shown in male chickens. (Key Words : Chicken, Cholesterol, Glutamic Acid,
Natto, Soybean)
* This research was supported by a research-aid fund of research
projects for utilizing advanced technologies in agriculture,
forestry and fisheries, Japan.
** Corresponding Author: Y. Miyaguchi. Tel: +81-29-887-1261,
Fax: +81-29-888-8525, E-mail: miayguti@mx.ibaraki.ac.jp
1 College of Agriculture, Ibaraki University, Ibaraki, 300-0393,
Japan.
2 National Institute of Livestock and Grassland Science, Ibaraki,
305-0901, Japan.
Received November 12, 2007; Accepted March 17, 2008

Fujiwara et al. (2008) Asian-Aust. J. Anim. Sci. 21(12):1766-1772
1767
chicken meat has recently become more popular in Japan
because of its better taste and firmer texture than broiler
meat. The chickens must be a Japanese species and have
been bred free-range. “Tsukuba-jidori” was developed in
Ibaraki Prefecture, Japan by crossbreeding between a male
White Cornish and a female crossbred chicken between
“Hinai-jidori” and Rhode Island Red.
To clarify the qualities of natto as a chicken feedstuff,
the effect of natto supplement on the productivity and
qualities of chicken meat were investigated in this study.
MATERIALS AND METHODS
Diets, birds, and conditions for feeding
Natto provided by Takano Foods Co. Ltd (Ibaraki,
Japan) was dried in an oven at 60°C for 60 h followed by
grinding into 1 mm fragments using a grinder. It was
determined that the resultant dried natto contained more
than 1×109 cfu/g. Forty newborn male or female Tsukuba-
jidori in each group (2 replicates of 5 chickens making a
subgroup) were randomly housed in 8 different cages. Each
of the 4 groups of chickens was assigned randomly; a basic
diet without natto was supplied to the control group.
Experimental diets containing a basic diet plus dried natto
at different dosages (1% and 2%) were supplied to the other
3 groups. For two groups, 1% and 2% natto supplemented-
diets were supplied for days 0 to 80 (1DN and 2DN). In the
remaining group, a basic diet without dried natto was
supplied for days 0 to 27 and subsequently the 2% natto
supplemented-diet was supplied for days 28 to 80 (2DN-A).
During the experiment, chickens were fed on an ad libitum
basis and had free access to water. Preparation of dried
natto according to the experimental design described above
is shown in Figure 1.
Preparation of meats and analytical procedure
After the feeding for 80 days, the chickens were
slaughtered by decapitation. Breast and bone-in thigh meat,
fillet, and abdominal fat were taken from the carcasses and
weighed after exsanguination. The thighbone was also taken
from the thigh and weighed, and the. Length of the bone
and thickness of cortical bone were measured by slide
gauge. About one-gram of cortical bone was pulverized and
incinerated to ash at 500°C for 2 h. The bone ash was
dissolved in HCl and the solution was used for the
measurement of calcium (Ca) and phosphorus (P). Calcium
was measured using an atomic absorption
spectrophotometer (AA6500F, Shimazu Co. Ltd., Kyoto,
Japan) and the phosphorus level was determined by a
colorimetric method. The pH value of the meat was
measured using a pH meter with a glass-electrode (Φ32PH
meter, Beckman, CA, USA). Meat samples were minced
and homogenized in 50 mM imidazole buffer. After
centrifugation, the water-soluble protein in the obtained
supernatant was measured by the Biuret method.
Furthermore, the supernatant was added to trichloroacetic
acid (TCA) and the amount of 10% TCA soluble peptides
was determined by the Folin method. Free glutamic acid
(Glu) in the meat was determined using an F-kit L-glutamic
acid (Roche Molecular Biochemicals, Germany). Briefly,
minced meat was dispersed in 1 mol/L perchloro acid and
homogenized with an IKA ultra disperser. After
Figure 1. Preparation of dried natto according to the experimental design explained in MATERIALS AND METHODS. Control, a basic
diet alone was supplied days 0 to 80. 1DN and 2DN, Basic diet with 1% and 2% dried natto was supplied for days 0 to 80, respectively.
2DN-A, Basic diet alone was supplied for days 0 to 27 and the diet with 2% dried natto was supplied for days 28 to 80.

Fujiwara et al. (2008) Asian-Aust. J. Anim. Sci. 21(12):1766-1772
1768
centrifugation, the resultant solution was added to 0.1 N
NaOH to reach pH 8.0 and adjusted to a volume of 25 ml
with water. The solution was used for the assay according to
the F-kit instruction manual.
Statistical analysis
All data were subjected to a 1-way ANOVA test.
RESULTS AND DISUCUSSION
Production performance
The effect of natto supplement on meat productivity is
shown in Table 1. The weight of the carcass was about 1.3
times higher in males than in females. The meat weights
obtained (breast, thigh, and fillet) showed the same
tendency as the whole carcass. The addition of 1% or 2%
natto did not influence on the meat productivity. The effect
of natto supplementation on thighbone growth is shown in
Table 2. All data including the length of the thighbone,
thickness of cortical bone, and the Ca/P ratio, were not
altered by natto supplementation. It is known that natto is
rich in isoflavones and vitamin K2 (menaquinone-7); the
former may help preventing bone loss by promoting
calcium absorption (Yamaguchi, 2002), and the latter acts
as a cofactor of gamma-carboxylase, which converts Glu
residues in osteocalcin molecules to gamma-
carboxyglutamic acid for promotion of normal bone
mineralization (Tsukamoto et al., 2000). The expected
results could not be obtained because of unsuitable
experimental conditions in this study though we regarded
the supplementation of natto should improve bone
metabolism.
Meat qualities
The effect of natto supplementation on the pH of meat is
shown in Figure 2. In male chickens, the pH of breast and
thigh meat decreased significantly in the 2% natto group
supplied for days 28 to 80 (2DN-A). This tendency was not
found in female chickens on the same diet. Various qualities
of meat depend on its pH. The nutritional composition of
breast and thigh meats such as moisture, crude protein (CP),
and fat content were investigated, showing that the protein
concentration of female thigh meat tended to decrease with
the supplementation of natto though the moisture and fat
concentrations of meat in males did not change significantly
(data not shown).
Many studies have identified relationships between feed
and meat qualities including color (Smith et al., 2002),
texture (Kristensen et al., 2002), and drip loss (Young et al.,
2004). However, effect of fermented soybean supplement
on the production performance and pH of meat has not been
investigated. Since the mechanism of the pH decrease by
Table 1. Effect of natto supplementation on meat production
Experimental groups
Cont. 1DN 2DN 2DN-A
Male (n = 10)
Carcass (g) 3,405 3,457 3,463 3,435
Breast meat, bone- in (g) 799 819 823 857
Thigh meat, bone-in (g) 918 889 921 917
Filet (g) 129.1 129.2 127.4 128.6
Thigh bone (right) (g) 30.5 29.7 30.3 31.6
Abdominal fat (g) 88.4 89.0 88.4 64.9
Female (n = 10)
Carcass (g) 2,538 2,495 2,510 2,504
Breast meat, bone-in (g) 637 616 603 620
Thigh meat, bone-in (g) 639 619 615 625
Fillet (g) 105.8 100.3 101.4 103.7
Thigh bone (right) (g) 19.6 18.6 19.0 18.1
Abdominal fat (g) 81.1 81.3 71.4 93.5
Cont., 1DN, and 2DN correspond to 0, 1, and 2% dried natto supplementation for days 0 to 80, respectively.
2DN-A, a basic diet was supplied for days 0 to 27 and subsequently 2% dried natto was supplied for days 28 to 80.
Table 2. Effect of natto supplementation on the bone growth of chicken
Experimental groups
Cont. 1DN 2DN 2DN-A
Thigh bone length (cm) 10.68±0.33 10.76±0.12 10.95±0.23 10.91±0.34
Cortical bone thickness (mm) 0.33±0.03 0.35±0.07 0.39±0.05 0.37±0.06
Ca/P ratio of the thigh bone 2.51±0.14 2.36±0.10 2.94±0.60 2.45±0.03
n = 3, Values are mean±SE.
Cont., 1DN, and 2DN correspond to 0, 1, and 2% dried natto supplementation for days 0 to 80, respectively.
2DN-A, a basic diet was supplied for days 0 to 27, and subsequently 2% dried natto was supplied for days 28 to 80.

Fujiwara et al. (2008) Asian-Aust. J. Anim. Sci. 21(12):1766-1772
1769
natto supplementation was not clarified in this study, we
intend to study the relationship between pH and
physicochemical properties of the meat of natto fed
chickens in our future research.
The water-soluble fractions such as the protein and
peptides contents in meat increased with the
supplementation of natto (Table 3). In male chickens, the
water-soluble protein content of thigh meat was
significantly higher in the 2DN-A treatment than in the
control. In female chickens, the water-soluble protein
content was not altered by natto supplementation. The
TCA-soluble peptide content male thigh meat as higher in
2DN than in the control (p<0.05). The free amino acids and
peptides that increase during post-mortem aging play an
important role in the formation of meat taste. It is reported
that the increase in free amino acids during the post-mortem
storage of meat is caused by the action of aminopeptidases
(Migita and Nishimura, 2006). Therefore, the free Glu
contents in the breast and thigh meats after feeding of natto
were determined, showing that the free Glu content of thigh
meat was higher in 2DN than in the control regardless of
sex (Figure 3). 2DN also increased the free Glu content in
female thigh meat. This tendency was not shown in breast
meat. Some di- or tri-peptides containing Ala, Asp, Val, Glu,
Ser, and Pro were recognized to enhance the taste of 0.02%
5'-inosinic acid (IMP) (Maehashi et al., 1999). Fujimura et
al. (2001) identified three compounds, free Glu, IMP, and
potassium ions, as active taste components in chicken meat
extracts. Glutamic acid and IMP, called “umami” taste, are
preferred by consumers and constitute a characteristic taste
of chicken meat. It is believed that free Glu is the most
important ingredient among these three active taste
compounds. Aging of meat also enhanced the increase in
several free amino acids.
It was believed that active taste components were not
influenced by diet (Farmer, 1999). However, it was recently
reported that free Glu and sensory scores in meat were
increased by a high CP diet, and the free Glu content was
increased by elevated dietary CP levels for 3 to 10 days
using Cobb strain female broilers (Fujimura and Kadowaki,
2006). Furthermore, the free Glu content was significantly
increased by dietary leucine (Leu). In particular, compared
Table 3. Effect of natto supplementation on soluble protein and peptide levels in chicken
Experimental groups
Cont. 1DN 2DN 2DN-A
Soluble protein (g/100 g)
Breast (male) 9.48±0.15 9.93±0.29 9.58±0.22 9.80±0.01
Breast (female) 11.5±0.55 12.3±8.8 12.5±0.17 12.4±0.12
Thigh (male) 3.94±0.11 4.41±0.18 4.25±0.06* 4.88±0.29**
Thigh (female) 5.92±0.15 5.71±0.09 5.91±0.19 5.87±0.31
TCA-soluble peptide (μmol/100 g)
Breast (male) 118.8±10.6 115.6±2.7 125.7±12.3 127.5±9.1
Breast (female) １04.5±2.8 107.2±4.9 115.6±3.1 109.0±8.3
Thigh (male) 84.6±5.5 103.9±8.3 129.2±19.1* 96.5±15.6
Thigh (female) 96.5±10.1 84.3±7.9 95.6±10.2 92.5±11.9
n = 3, Values are mean±SE.
Cont., 1DN, and 2DN correspond to 0, 1, and 2% dried natto supplementation for days 0 to 80, respectively.
2DN-A, a basic diet was supplied for days 0 to 27, and subsequently 2% dried natto was supplied for days 28 to 80. * p<0.1, ** p<0.05.
Breast
Thigh
5.6
5.7
5.8
5.9
6.0
6.1
6.2
5.6
5.7
5.8
5.9
6.0
6.1
6.2
♂♀
Cont. 1DN 2DN 2DN-A
B
pH
pH
AB AB A AB AB AB A
BB ABA A A A A
Breast
Thigh
5.6
5.7
5.8
5.9
6.0
6.1
6.2
5.6
5.7
5.8
5.9
6.0
6.1
6.2
♂♀
Cont. 1DN 2DN 2DN-A
B
pH
pH
AB AB A AB AB AB A
BB ABA A A A A
Figure 2. Effect of the supplementation of natto on the pH of the
breast and thigh meat. Cont., 1DN, and 2DN correspond to 0, 1,
and 2% dried natto supplementation for days 0 to 80, respectively.
2DN-A, a basic diet was supplied for days 0 to 27, and
subsequently 2% dried natto was supplied for days 28 to 80.
Vertical bar, standard error (n = 5). Data bearing the same
superscripts are not significantly different (p>0.05).

Fujiwara et al. (2008) Asian-Aust. J. Anim. Sci. 21(12):1766-1772
1770
with the Leu 130% group, free Glu was increased by 17%
in the Leu 100% group (Imanari et al., 2007).
In this study, the increase in free Glu in meat was shown
by 2% natto supplementation though the 2% dried natto diet
did not have a high level of CP or Leu. It is reported that
Bacillus as a probiotic enhanced not only intestinal flora but
also meat production and qualities and lipid metabolism
(Santoso et al., 1995; Cavazzoni et al., 1998). Several
researchers have shown previously that the peptide content
of soy-fermented products is greater than that of
unfermented soybeans (Okamoto et al., 1995). Various
reports suggested that fermentation increased protein
content, eliminated trypsin inhibitors, and reduced the
peptide size in soybeans, soybean meal, and fermented
soybean might be of more benefit to livestock as a novel
feed ingredient (Hong et al., 2004). Therefore, natto
supplementation could enhance the meat qualities not by
providing additional protein but by other factors such as
biofunctional peptides in natto and Bacillus natto as a
probiotic.
Meat cholesterol levels of chickens fed with natto
supplement are shown in Figure 4. The cholesterol content
of thigh meat was higher in females than in males. The
supplementation of natto decreased meat cholesterol in
female thigh meat significantly (p<0.05). The same
tendency was shown in breast meat. On the other hand,
significant differences were not found in males. It is
reported that the supplementation of red mold rice
decreased the serum and meat cholesterol levels of broiler
chickens (Wan et al., 2005). It was also reported that
feeding of high levels of copper reduced the cholesterol
content by approximately 25% in the edible muscle tissue
of broiler chickens for 42 d without altering the growth of
the chickens or substantially increasing the copper content
of the edible meat (Bakalli et al., 1995). The high molecular
weight fraction of soybean protein hydrolyzates showed
hypocholesterolemic effect in rats (Sugano et al., 1989).
In the studies of bacterial cultures, egg with lower
cholesterol levels in the yolk were produced by feeding of a
dried culture of Bacillus subtilis, which is the same strain as
Bacillus natto (Xu et al., 2006). It is known that probiotics
in intestinal flora digest cholesterol for their own cell
metabolism. Gilliland (1985) reported that microorganisms
Breast
Thigh
0
0.01
0.02
0.03
0.04
0.05
0.06
0
0.01
0.02
0.03
0.04
0.05
0.06
♂♀
Cont. 1DN 2DN 2DN-A
Free glutamic acid (g/100 g)
**
*
Free glutamic acid (g/100 g)
*
Breast
Thigh
0
0.01
0.02
0.03
0.04
0.05
0.06
0
0.01
0.02
0.03
0.04
0.05
0.06
♂♀
Cont. 1DN 2DN 2DN-A
Free glutamic acid (g/100 g)
**
*
Free glutamic acid (g/100 g)
*
Figure 3. Effect of the supplementation of natto on the free
glutamic acid concentration in the breast and thigh meat. Cont.,
1DN, and 2DN correspond to 0, 1, and 2% dried natto
supplementation for days 0 to 80, respectively. 2DN-A, a basic
diet was supplied for days 0 to 27, and subsequently 2% drie
d
natto was supplied for days 28 to 80. Vertical bar, standard error (n
= 3). * and ** indicate that the values are significantly different at
p<0.1 and p<0.05, respectively.
Figure 4. Effect of the supplementation of natto on the cholesterol
content in the breast and thigh meat. Cont. and 2DN correspond to
0 and 2% dried natto supplementation for days 0 to 80,
respectively. Vertical bar, standard error (n = 3). ** Indicate tha
t
the values between Cont. and 2DN are significantly different at
p<0.05.
0
20
40
60
80
100
0
20
40
60
80
100
♂♀
Cont. 2DN
**
**
Cholesterol (g/100 g meat)
Breast
Thigh
Cholesterol (g/100 g meat)
0
20
40
60
80
100
0
20
40
60
80
100
♂♀
Cont. 2DN
**
**
Cholesterol (g/100 g meat)
Breast
Thigh
Cholesterol (g/100 g meat)

Fujiwara et al. (2008) Asian-Aust. J. Anim. Sci. 21(12):1766-1772
1771
absorb cholesterol and cause a decrease in cholesterol in
host animals. Fukushima and Nakano (1995) suggested that
the serum cholesterol decrease in host animals fed
probiotics was caused by inhibition of HMG-CoA reductase
activities. Haddin et al. (1996) showed that yolk cholesterol
levels decreased by feeding of 4×108 million living
Lactobacillus/g diet for 48 week.
However, our results showed that the decrease in meat
cholesterol level was found in females specifically,
suggesting that natto components act as female hormones
such as isoflavones with estrogen-like functions. Kishida et
al. (2006) reported that dietary isoflavone-rich fermented
soybean extract decreased the serum cholesterol
concentrations in female rats but did not affect the
concentrations in male rats. Furthermore, in accordance
with the deglycosylation of isoflavone glycosides, the
estrogenic activity of black soymilk by Bacillus natto on the
ERβ estrogen receptor increased threefold (Kuo et al.,
2006). The meat cholesterol lowering effect of natto
requires further study.
Recently, it has been well-shown that bioactive peptides,
which possess diverse and unique health benefits including
the prevention of age-related chronic disorders, such as
cardiovascular disease, cancer, obesity, and decreased
immune function, are produced by fermented soybeans in
human diets (Mejia and De Lumen, 2006). In the poultry
industry, the supplementation of probiotics has recently
attracted interest from the point of poultry health.
Antibiotics are in widespread use to prevent poultry
pathogens and disease so as to improve meat and egg
production. However, the continued use of dietary
antibiotics has resulted in common problems, such as the
development of drug resistant bacteria (Sorum and Sumde,
2001), imbalance of normal microflora (Andermont, 2000),
and drug residues in the body (Burgat, 1991). As a result of
these problems, it has become necessary to develop
alternatives using beneficial microorganisms. A probiotic is
a live microbial feed supplement that beneficially affects
the host animal by improving its intestinal microbial
balance (Fuller, 1989), and is recommended as an effective
alternative to antibiotics (Sissons, 1989). Since it is possible
that natto, which contains bioactive peptides and living
Bacillus natto as a probiotic for poultry, we are also
investigating the immunomoduratory effects of natto from
the point of view of animal health.
ACKNOWLEDGMENTS
We are grateful to the manager of Takano Foods Co.
Ltd., Ken-Ichiro Kaneko for providing fermented soybean
products. We wish to thank Mr. Kazuo Namai of the
Laboratory of Poultry, Ibaraki Prefectural Livestock
Research Center.
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