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Food Sci. Biotechnol.

21(5): 1397-1403 (2012)


DOI 10.1007/s10068-012-0184-0

RESEARCH ARTICLE

Effects of Fermented Black Soybean Pulp on Lipid and


Bone Metabolism in Ovariectomized Rats
Go-Eun Hong, Chang-Won Pyun, Soo-Ki Kim, Kyu-Ho Han, Cheul-Young Yang, and Chi-Ho Lee

Received: 29 March 2012 / Revised: 29 May 2012 / Accepted: 14 June 2012 / Published Online: 31 October 2012
© KoSFoST and Springer 2012

Abstract To investigate the effect of fermented black Introduction


soybean pulp on serum lipid metabolism and osteoporosis
in ovariectomized rats, Sprague-Dawley rats were divided Improving economic standards and developments in
into 4 groups: sham-operated control group, ovariectomized medical technology have caused increased life expectancy.
control group, ovariectomized group treated with black The average life span of Korean women has increased
soybean pulp (BSP), and ovariectomized group treated steadily from 80.45 years in 2002 to 83.77 years in 2009,
with fermented black soybean pulp (FBSP). Rats were fed according to the report by the Korean National Statistical
experimental diets for 8 weeks after ovariectomy. Compared Office. This value is higher than the average lifespan of
to the control, FBSP supplemen-tation led to the improving 79.5 years reported by the Organization for Economic Co-
effect for femur weight, abdominal fat, alanine amino- operation and Development (OECD) (1,2). Female menopause
transferase, aspartate aminotransferase activity, hepatic lipid is a stage of life that usually starts at 45-50 years; thus,
levels, and thiobarbituric acid reactive substances. Also, many women live over 1/3 of their lives after menopause.
serum HDL-cholesterol, estradiol levels, serum alkaline Menopause is a result of ovarian senescence, and the
phosphatase activity, and osteocalcin levels in the FBSP consequent decrease in estrogen production in the ovaries
group were significantly lower than in the control group affects bone and lipid metabolism and increases the rates of
(p<0.05). These results suggest that FBSP is effective in osteoporosis, obesity, and cardiovascular disorders (3).
improving lipid metabolism and preventing osteoporosis in Osteoporosis in postmenopausal women is caused by
ovariectomized rats. estrogen deficiency, which leads to increasing bone loss
with accelerated bone resorption and inhibition of calcium
Keywords: isoflavone, aglycone, black soybean pulp, absorption in the colon (4). The risk of fracture due to
fermentation, ovariectomy osteoporosis in postmenopausal women over 50 years of
age is approximately 40%, which is a serious concern (5).
Hormone replacement therapy (HRT) has been used to
prevent menopausal diseases, including osteoporosis, but
Go-Eun Hong, Chang-Won Pyun, Chi-Ho Lee () side effects such as increasing rates of cardiovascular
Department of Food Science and Biotechnology of Animal Resources,
Konkuk University, Seoul 143-701, Korea diseases, breast cancer, and uterine cancer (6,7) have been
Tel: +82-2-450-3681, Fax: +82-2-453-1948 reported. Therefore, there has been increasing interest in
E-mail: leech@konkuk.ac.kr
natural phytoestrogens which have similar structure and
Soo-Ki Kim biological function to estrogen (8). Isoflavones are phenolic
Department of Animal Science and Environment, Konkuk University,
Seoul 143-701, Korea
compounds from soybeans that exist as glycosides (daidzin
and genistin) or aglycones (daidzein and genistein).
Kyu-Ho Han Isoflavones generally exist in the glycoside form (9), but
Department of Food Science, Obihiro University of Agriculture and
Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan during fermentation, microbial enzymes break the carbohydrate
bond by activity of β-glucosidase and increase the
Cheul-Young Yang
Department of Food Science and Service, Eulji University, Seongnam, concentration of aglycone (10,11). Compared to the
Gyeonggi 461-713, Korea glycoside form, the aglycone isoflavone form has a higher
1398 Hong et al.

rate of absorption in the small intestine as well as higher Table 1. Composition of experimental diets (g/kg diet)
antioxidant activity (12,13). Hence, fermented soy foods Ingredient AIN-93M BSP1) FBSP2)
containing larger amounts of aglycone isoflavones are Casein 140.00 131.72 138.13
more effective in osteoporosis or other menopausal disorders. Sucrose 100.00 100.00 100.00
Isoflavones are known for their anticancer activity and Dextrose 155.00 155.00 155.00
control of cell cycle and growth (9). They also have preventive Corn starch 465.692 437.392 460.992
or therapeutic effects in hypertension, cardiovascular Cellulose 50.00 40.83 48.71
disorders, and hormone-associated diseases, especially Soybean oil 40.00 21.78 36.33
postmenopausal syndrome (14-17). The isoflavone content Black soybean pulp 0 64.02 0
of soybeans varies by type, harvest year, and production Fermented black soybean pulp 0 0 11.52
region (18). Black soybeans have high isoflavone TBQH3) 0.008 0.008 0.008
concentrations and physiological activity and have been Minerals (Ca, P free) 35 35 35
used as a folk medicine for their pharmacological effects Vitamin mix 10 10 10
(19,20). In addition, black soybean pods have a high level L-Cysteine 1.8 1.8 1.8
of anthocyanin, a powerful antioxidant (21). There is a Choline bitartrate 2.5 2.5 2.5
recent focus on the use of black soybean foods such as Total 1,000.0 1,000.0 1,000.0
black tofu or black soymilk for health benefits. Soy pulp is Crude protein 141.80 141.80 141.80
produced as a byproduct during soybean food processing. Crude carbohydrate 40.00 40.00 40.00
Sometimes, soy pulp is used as animal feed, but it is mostly Crude fiber 50.00 50.00 50.00
1)
buried as waste (22). There is a worldwide increase in BSP, black soybean pulp added into AIN-93M diet without Ca2+
demand for functional health foods, such as those that and P
2)
FBSP, fermented black soybean pulp by L. acidophilus added into
contain isoflavones known for their effects on hormone AIN-93M diet without Ca2+ and P
3)
deficiency-associated diseases and in the prevention of TBQH, tert-butylhydroquinone (antioxidant)
osteoporosis in postmenopausal women; however, isoflavone
has a low yield compared to its price and is difficult to Temperature and humidity were kept at 23±2oC and
distribute (23). In this study, black soybean pulp fermented 55±5%, respectively, with room lighting on a 12 h light/
by Lactobacillus acidophilus was investigated the effect on dark cycle. The compositions of the experimental diets are
serum lipid metabolism and osteoporosis in ovariectomized shown in Table 1. The Con and Sham groups were fed a
(OVX) rats. purified control diet, and the BSP and FBSP groups were
fed the same control diet with lyophilized black soybean
pulp and fermented black soybean pulp added to 64.02 and
Materials and Methods 11.52 g/kg of feed, respectively. BSP and FBSP were
produced in accordance with a previous study (24). The
Black soybean pulp production Black soybeans (Korean quantity of BSP and FBSP added to the diet was based on
cultivar; ‘Seoritae’) were purchased at a local market in a previously published paper and on the standard of
Hwayang-dong, Seoul, Korea. Black soybeans were soaked Korean Food & Drug Administration (KFDA 2010-7, soy
in a 3:1 ratio of water to black soybeans for 8 h, and then isoflavone) of 25 mg/day. In a previous study, the isoflavone
steamed. Black soybean pulp was then produced by aglycone contents of BSP and FBSP were found to be 0.78
grinding the beans, removing the water with a cotton cloth, and 4.34 mg/100 g dried powder, respectively (24). Based
and freeze-drying. on these results, the appropriate amounts of BSP and FBSP
were added to the diet. All animals were fed a Ca2+- and P-
Animal experimental design Twenty-four sexually mature deficient purified rodent diet (Dyets, Bethlehem, PA, USA)
female Sprague-Dawley rats (8 weeks of age) were purchased with water ad libitum. All animal experiments were approved
from Central Laboratory Animal Inc. (Seoul, Korea). Six by the Institutional Animal Care and Use Committee
rats were randomly assigned to laparotomy as sham- (IACUC, Approval no. KU11089). Food intake was
operated controls (Sham) and the remaining 18 rats to recorded daily and body weight was measured weekly. The
bilateral ovariectomy. One week after surgery, the OVX food efficiency ratio was calculated as total weight gain −
rats were divided into 3 groups: OVX with normal diet total food intake. At the end of the 8-week treatment
(Con), OVX with normal diet containing black soybean period, the rats were fasted for 24 h and anesthetized under
pulp supplement (BSP), and OVX with normal diet appropriate conditions. Blood was collected from the
containing fermented black soybean pulp supplement abdominal aorta and the serum was separated by
(FBSP). All animals were housed in an environmentally centrifugation (3,000×g, 20 min) after 1 h. The weights of
controlled laboratory (Konkuk University, Seoul, Korea). the liver, kidney, spleen, and abdominal fat were recorded
Effect of Fermented Black Soybean Pulp 1399

and the specimens were stored at −80oC before further Table 2. Body weight gain and feed efficiency in rats fed for 8
analysis. Muscle tissues, adipose tissues, and ligaments weeks
were removed entirely from both sides of the femur, Group1) Body weight gain (g) Feed efficiency (%)
a2)
weighed, and then fixed in 10% formalin prior to analysis. Con 209.7±15.53 10.0±0.59a
Sham 089.7±10.07b 4.8±0.37b
Experimental analysis BSP 209.3±2.08a 9.8±0.38a
Biochemical parameters in serum: Levels of total FBSP 215.3±28.57a 9.9±0.92a
cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, 1)
Con, control, ovariectomized; Sham, sham-operated; BSP,
aspartate aminotransferase (AST), alanine aminotransferase ovariectomized, black soy pulp; FBSP, ovariectomized, fermented
black soy pulp
(ALT), Ca2+, and P concentrations in serum were measured 2)
Values are mean±SD of 6 rats in each group after 8 weeks; Values
by Biotoxtech Co. (Chungwon, Korea). The atherogenic with different superscripts within a column are significantly
index (AI) was calculated by the Haglund method (25) as different (p<0.05).
(total cholesterol−HDL-cholesterol)/HDL-cholesterol.
Osteocalcin in serum: The osteocalcin concentration in at 3,000×g for 10 min. The supernatant was collected and
serum was analyzed by the sandwich enzyme-linked the absorbance was measured using a spectrophotometer
immunosorbent assay (ELISA) method using the rat (Optizen 2120UV; Mecasys Co., Ltd., Daejeon, Korea) at
dsteocalcin EIA kit (Biomedical Technologies Inc., 535 nm. The results were calculated using a standard curve
Stoughton, MA, USA). A 7-stage osteocalcin standard made with 1,1,3,3-tetraethoxypropane (Sigma-Aldrich, St.
curve was prepared, with concentrations from 0.78 to 50 Louis, MO, USA) as measured by malondialdehyde
ng/mL. Experiments were carried out in duplicate and the production.
results are presented as averages. Absorbance was measured
by an ELISA microplate spectrophotometer (Biotek Inc., Statistical analysis The results are presented in mean±
Winooski, VT, USA) at 450 nm within 15 min after the standard error (SE) and were analyzed using SAS (Statistic
addition of stop solution. Analysis System, Cary, NC, USA) ver. 8.2 software. Duncan’s
Estradiol in serum: The estradiol concentration in serum multiple range test was used to determine which means
was analyzed using the mouse/rat estradiol ELISA kit were significantly different (p<0.05).
(Calbiotech Inc., Spring Valley, CA, USA) and absorbance
was measured using an ELISA microplate spectrophotometer
(Biotek Inc.) at 450 nm within 15 min after the addition of Results and Discussion
stop solution. A 5-stage estradiol standard curve was prepared,
with concentrations from 3 to 300 pg/mL. The analysis Body weight gain and food efficiency The results were
was performed in duplicate and results are presented as calculated in terms of change in body weight 2 weeks after
averages. ovariectomy and at the end of the 8-week treatment period.
Lipid profile in liver: Sample extraction for hepatic total Body weight gain was higher in every group than that in
lipid level analysis was done using the modified Folch the Sham group. This result is similar to those of other
method (26). Briefly, 2 mL of 0.88% NaCl was added to previous reports that recorded weight gain after the surgical
300 mg of liver tissue. The tissue was homogenized 3 times removal of ovaries in rats (28,29). In addition, Okasaki et al.
under ice-cold conditions with 2 mL of chloroform+ (30) reported that estrogen promotes osteoblast differentiation
methanol (2:1 v/v). The extracted liquid was collected in a but inhibits adipocyte differentiation.
15-mL test tube and centrifuged at 3,000×g for 20 min.
The upper phase was removed as completely as possible Organs The organ weights of the experimental animals
and the lower phase remained in the test tube. The liquid are shown in Table 3. Kidney weight was not significantly
was evaporated using pure nitrogen gas. After evaporation, different after treatment (p<0.05). The elevated spleen
the tube was weighed and the total lipid content in the liver weights in OVX rats were consistent with enhanced post-
was estimated. Thiobarbiturate (TBA) values in the liver surgical immune response. The right femur weight was
were determined by the Ohkawa method (27) with minor significantly lower in the Con group compared to the other
modifications. In brief, 300 mg of liver tissue was groups (p<0.05), and the values of the BSP and FBSP
homogenized with 1.5 mL of 0.05 M phosphate buffer (pH groups were significantly higher, as was that of the Sham
7.4) under ice-cold conditions and 0.5 mL aliquots were group (p<0.05). Ca2+- and P-free diets were used to
collected in triplicate, after which 2 mL of 0.67% TBA+ minimize the effect of feed, and this result indicated that
acetic acid (1:1 v/v) was added. The mixture was heated in the isoflavones in BSP and FBSP had a positive effect on
a boiling water bath to 95oC for 50 min. After heating, 5 femur preservation. The amount of abdominal fat was
mL of butanol was added and the mixture was centrifuged significantly higher in the Con group than in the Sham
1400 Hong et al.

Table 3. Weights of organs from rats fed for 8 weeks (unit: g)


1)
Group Kidney Spleen Femur (R) Abdominal fat
a2) a b
Con 1.68±0.065 0.64±0.034 0.91±0.244 17.65±3.082a
Sham 1.48±0.147b 0.51±0.028c 1.01±0.042a 08.21±0.950c
BSP 1.67±0.146ab 0.57±0.042b 1.05±0.040a 15.57±1.899ab
FBSP 1.62±0.098ab 0.66±0.013a 1.03±0.031a 14.75±2.524b
1)
Con, control, ovariectomized; Sham, sham-operated; BSP, ovariectomized, black soy pulp; FBSP, ovariectomized, fermented black soy pulp
2)
Values are mean±SD of 6 rats in each group after 8 weeks; Values with different superscripts within a column are significantly different
(p<0.05).

Table 4. Biochemical parameters of the serum of rats


Group1) AST (U/L) ALT (U/L) TC (mg/dL) TG (mg/dL) LDL-C (mg/dL) HDL-C (mg/dL) AI
a a b NS a b
Con 165.78±19.034 54.58±8.437 81.25±3.096 15.50±2.082 4.98±0.655 22.98±1.162 2.536
Sham 124.85±7.471b 43.88±2.004a 75.50±2.517b 13.00±3.162 3.20±0.497b 24.80±1.503b 2.044
BSP 159.63±18.602a 43.40±8.545a 97.50±4.509a 12.25±1.893 5.30±0.424a 28.50±0.876a 2.421
FBSP 117.28±7.692b 31.15±5.588b 85.00±18.833ab 11.50±4.435 4.55±1.215a 27.83±3.117a 2.055
1)
Con, control, ovariectomized; Sham, sham-operated; BSP, ovariectomized, black soy pulp; FBSP, ovariectomized, fermented black soy pulp
2)
Values are mean±SD for 6 rats in each group after 8 weeks; Values with different superscripts within a column are significantly different
(p<0.05); NS, not significant

group (p<0.05). The amount of abdominal fat was Table 5. Effect of dietary black soybean pulp and fermented
significantly lower in the FBSP group than in the Con black soybean pulp on serum alkaline phosphatase (ALP)
activity, Ca2+, and P concentrations
group; it was even lower, although not significantly so, in
the BSP group. Group1) ALP (U/L) Ca (mg/dL) P (mg/dL)
a2) a
Con 522.73±68.375 10.18±0.310 5.44±0.288b
Biochemical parameters in serum The results of Sham 463.70±23.927b 10.10±0.346a 5.88±0.266b
biochemical measurements in serum are shown in Table 4. BSP 443.63±24.160b 09.60±0.316b 6.74±0.465a
Serum AST and ALT activities were used as markers of FBSP 459.88±12.036b 009.88±0.126ab 6.04±0.461b
liver damage since AST and ALT are detected in blood 1)
Con, control, ovariectomized; Sham, sham-operated; BSP,
after exocytic release upon liver damage. As shown in ovariectomized, black soy pulp; FBSP, ovariectomized, fermented
black soy pulp
Table 4, AST and ALT activities in the FBSP group were 2)
Values are mean±SD of 6 rats in each group after 8 weeks; Values
significantly lower than those in the other groups (p<0.05). with different superscripts within a column are significantly
different (p<0.05).
The BSP group showed a significantly higher level of total
cholesterol than did the Con and Sham groups (p<0.05).
The LDL-cholesterol level in the BSP group was not in serum with increased calcium release from bone (32). In
significantly different, but the HDL-cholesterol level was this study, serum calcium levels in the BSP group were
significantly higher than that of the Con group (p<0.05). In significantly lower than those of both the Con and Sham
other words, the higher total cholesterol level of the BSP groups (p<0.05). Calcium levels in the FBSP group tended
group was a consequence of high HDL-cholesterol content. to be lower but not significantly so (p<0.05). The serum
The HDL-cholesterol level in the FBSP group was phosphorus level was significantly higher in the BSP group
significantly higher than that in the Con group (p<0.05). than in the other groups (p<0.05), and tended to be higher,
Estrogen is known for preventing arteriosclerosis and but not significantly so, in the FBSP group. This result is
cardiovascular diseases by decreasing LDL-cholesterol and similar to another previous report (33) wherein serum
increasing HDL-cholesterol (31). The AI in the FBSP phosphorus in OVX rats was higher than that in normal
group was lower than that in the Con group. This result rats. In addition, a previous study reported that isoflavone-
indicated that FBSP has a positive effect on lipid containing nijiru depletes serum phosphorus concentration
metabolism in the blood. Triglyceride levels were not in both men and women (34), while Kim and Lee (35)
significantly different (p<0.05). Serum alkaline phosphatase reported that the phosphorus level in serum is not related to
(ALP), Ca2+, and P levels are shown in Table 5. Serum ovariectomy. Overall, serum calcium and phosphorus
Ca2+ and P levels were normal in all groups, with ranges of reactions for bone metabolism are complex and require
9.60-10.18 and 5.44-6.74 mg/dL, respectively. An increase further investigation. Serum ALP, a bone formation marker,
of bone loss and fracture risk has been reported due to was reported to increase after oophorectomy (36). Likewise,
increased bone turnover and parathormone concentration in this study, ALP activity in the Con group was
Effect of Fermented Black Soybean Pulp 1401

Fig. 1. Effect of dietary black soybean pulp and fermented Fig. 2. Effect of dietary black soybean pulp and fermented
black soybean pulp on serum osteocalcin concentrations. Con, black soybean pulp on serum estradiol (E2) concentration.
control, ovariectomized; Sham, sham-operated; BSP, ovariectomized, Con, control, ovariectomized; Sham, sham-operated; BSP,
black soy pulp; FBSP, ovariectomized, fermented black soy pulp. ovariectomized, black soy pulp; FBSP, ovariectomized, fermented
Values with different superscripts are significantly different black soy pulp. Values with different superscripts are significantly
(p<0.05). different (p<0.05).

significantly higher than in the Sham group, but was not Table 6. Hepatic lipid levels and levels of thiobarbituric acid
significantly so in the BSP and FBSP groups compared to reactive substances (TEARS) in the experimental groups
the sham group (p<0.05). This result indicates that Total lipid TBARS
Group1) Liver (g)
isoflavones from BSP and FBSP have positive effects on (%/liver) (MDA mg/kg)
the bone metabolism in ovariectomized rats as an estrogen Con 7.61±0.249a2) 1.3±0.27a 1.0±0.14a
like substance. Sham 7.20±0.436ab 0.4±0.18b 0.9±0.07ab
BSP 7.34±0.344ab 1.0±0.44ab 0.9±0.06ab
Serum osteocalcin level Serum osteocalcin level, an FBSP 7.00±0.191b 0.8±0.13b 0.8±0.14b
indicator of bone formation, was measured. Serum ALP, 1)
Con, control, ovariectomized; Sham, sham-operated; BSP,
usually used as a bone formation marker, is less sensitive ovariectomized, black soy pulp; FBSP, ovariectomized, fermented
black soy pulp
in indicating subtle bone changes because it is also 2)
Values are mean±SD of 6 rats in each group after 8 weeks; Values
synthesized in the liver and kidney (37). Osteocalcin is a with different superscripts within a column are significantly
different (p<0.05).
tiny noncollagenous protein with a molecular weight of 4.9
kDa which is secreted by osteoblasts. It contains γ-
carboxyglutamic acid, the only molecule that can bind to and FBSP groups were significantly higher than those in
calcium, and exists specifically in bone dentin. Moreover, the Con group (p<0.05).
it combines with the bone extracellular matrix and some of
these complexes are isolated from blood (38). Increased Lipid profile in liver Hepatic lipid levels and TBA-
serum levels of osteocalcin are seen in metabolic bone reactive substances (TBARS) levels were as shown in
diseases and bone reformation due to increased osteoblast Table 6. Hepatic lipid accumulation may cause oxidative
activity, as during rapid bone turnover (39). In the results stress and lipid peroxidation (40). Hepatic lipid levels in
shown in Fig. 1, osteocalcin levels in the Con group were the Con group were the highest (p<0.05), and tended to be
significantly higher than those in the Sham group for lower, but not significantly so, in the BSP group. Hepatic
oophorectomy. Osteocalcin levels in the FBSP group were lipid levels in the Sham and FBSP groups were
significantly lower than those in the Con group (p<0.05). significantly lower (p<0.05) than those in the Con group.
The BSP group had the lowest level of osteocalcin (p<0.05). TBARS levels were investigated to assess lipid peroxidation
This appears to be an effect of dietary supplementation in the liver and loss of physiological activity due to
with BSP and FBSP. estrogen deficiency following ovariectomy. TBARS levels
in the liver were significantly lower in the FBSP group
Estradiol levels in serum Estradiol is the main form of than in the Con group (p<0.05). TBARS levels tended to
the female sex hormone estrogen secreted by follicles, and be lower, but not significantly so, in the BSP group.
controls the female reproductive cycle. The serum estradiol Therefore, providing FBSP in the diet appears to suppress
level is rapidly decreased after ovariectomy. Serum estradiol the accumulation of fat or lipid peroxide in the hepatic
measurements after 8 weeks of treatment are shown in Fig. tissue. These results were caused by isoflavones in the
2. In the case of the Con group, serum estradiol levels were treatment, and are supported by the research of Lee et al.
significantly decreased by ovariectomy. Levels in the BSP (40), which showed that genistein decreased hepatic fat
1402 Hong et al.

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