1
BIOLOGICAL EVALUATION OF PROBIOTIC
FERMENTED MILK (RAYEB) ON OBESE RATS
Omnia G. Refaat ; Mohamed A. Arafa ; Naeem M. Rabeh and
Ranim S. Sabra
Nutrition and Food Science Dept., Home Economic Faculty, Helwan University
Key Words: fermented milk (Rayeb), vitamin D, obesity, weight, liver
and kidney function.
ABSTRACT
Probiotics have been widely explored in food processing,
production, research and disease prevention, therefore, the present study
was conducted to evaluate the biological evaluation of probiotic
fermented milk (Rayeb) on obese rats for 8 weeks. The rats (n=35) were
classified into 5 groups, as follows: Group (1): fed on basal diet and kept
as negative control group. While the rats (n= 28) were fed on high fat
diet for four weeks to induce obesity then were divided into four groups,
group (2) was fed on high fat diet only and served as control positive
group. Groups (3 and 4): obese rats were fed on high fat diet and were
given orally 1 ml of fermented milk “Rayeb” and Vitamin D at 600
IU/Kg BW respectively. Group (5): was fed on basal diet and was given
orally a mixture of (1 ml of Rayeb and Vitamin D at 600 IU/Kg BW).
The results indicated that, the supplementation with fermented
milk (Rayeb), vitamin D or their mixture significantly decreased
(P<0.05) the final body weight compared to the positive control group by
26.27%, 21.84%, 30.03%, respectively due to lowering the feed intake
and the feed efficiency ratio of the obese rats. Moreover, the
concentration of leptin hormone as well as the percent of peritoneal fat
bad were significantly lowered (P<0.05) for all treated groups. It was
observed that, the supplementation with the tested food materials
improved the liver and kidney functions as well as lipid profile of the
obese rats. It could be concluded that, fermented milk (Rayeb), vitamin D
or their mixture might be useful in weight reduction.
INTRODUCTION:
The prevalence of obesity has reached epidemic proportions over
the last few decades. In 2013, 36.9% of adult (age ≥20 years) men and
29.8% of women were considered overweight (body mass index [BMI]
25–29.9 kg m2 ) or obese (BMI ≥30 kg m2 ) (Ng et al., 2014), and recent
trend analyses show that the number of subjects who are overweight or
obese is continuing to rise worldwide (NCDRFC, 2016). According to
Egypt. J. of Appl. Sci., 35 (9) 2020 85-102
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the World Health Organization (WHO, 2000), in 2035, 39% of people in
today‟s society will be affected by obesity. That is why there is now an
increased need to create new public policies in prevention and in primary
health care (Khayatzadeh-Mahani et al., 2018). Because of the
multifaceted nature of obesity, there is no single or simple solution to
combat this growing epidemic. Novel, and most likely individualized
interventions, may thus be necessary to effectively prevent and treat
overweight and obesity (Borgeraas et al., 2018).
Probiotics are defined as “live microorganisms which, when
administered in adequate amounts, confer a health benefit on the host”
(Food and Agriculture Organization, 2006). Probiotic products are
available in many different forms worldwide, including pills, powders,
foods, and infant formula (Sanders, 2008). In the meantime, emerging
evidence reveals a large number of microorganism genera, such as
Lactobacillus, Bifidobacterium, Saccharomyces, Streptococcus, and
Enterococcus, whose supplementation in the diet might play a role in
prevention or management of obesity (De Vrese and Schrezenmeir ,
2008 and Morelli, and Capurso, 2012).
By-products from the bacterial fermentation process might lower
appetite and increase satiety (Cani et al., 2009), the microbiota may
suppress diet-induced obesity through increased energy expenditure
(Watanabe et al., 2012). Interestingly, obesity, as compared with normal
weight, is associated with a disease-specific dysbiotic shift in the faecal
microbiota and also a lower bacterial richness (Chatelier et al., 2013).
Energy restriction and weight loss, on the other hand, is associated with
an increased bacterial richness (Cotillard et al., 2013).
Oral administration of viable strains of bacteria (probiotics) has
been proposed as a way of manipulating the gut ecosystem to flavor
weight reduction or decrease weight gain; however, the mechanisms by
which probiotic supplementation may influence the gut microbiota are
largely unknown (Sanders, 2016)
Pereira-Santos et al., (2015) suggested that there are several
possible vehicles for the relationship between 25(OH)Vit D and weight
loss. The lack of 25(OH)Vit D status has been thought to be a possible
reason behind higher adiposity through the regulation of parathyroid
hormone (PTH) and modulation of adipogenesis The meta-analysis lays
the foundation for defining the potential clinical efficacy of vitamin
D supplementation as a potential therapeutic option for weight loss
programs (Perna, 2019).
86 Egypt. J. of Appl. Sci., 35 (9) 2020
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Therefore, the present study was conducted to evaluate the
biological evaluation of probiotic fermented milk “Rayeb" with Vitamin
D (VD) on obese rats.
MATERIALS AND METHODS
Materials: Fermented milk (Rayeb) Natural Cow's Milk containing
mixture of three strains of probiotics (Lactobacillus acidophilus,
Bifidobacterium bifidum, and Streptococcus thermophilus) at the level
of 107 CFU/ml was purchased from the local market. The viabilities of
the administered strains were confirmed.
Rats: A total of 35 adult male albino rats, Sprague Dawley strain,
weighing (150 g) were obtained from the Laboratory Animal Colony,
Helwan, Cairo.
Chemicals: Casein, cellulose, vitamins and minerals were obtained from
Morgan Company for Commerce and Chemicals. Capsules of
Cholecalciferol (Vitamin D3) at the concentration of 1000 IU were
purchased from local pharmacy. Starch was obtained from the Egyptian
Starch and Glucose, Mostorod Factory, Qalyubia, Egypt. Corn oil, Beef
tallow and Sucrose were obtained from local market. Kits for blood
analysis were purchased from Gama Trade Company for Chemicals,
Cairo, Egypt.
Microbial media: The microbial media used throughout the study had
been purchased from Difco and Oxoid.
Methods: The biological experiment and the chemical analysis were
carried out at the Post Graduated Lab, Faculty of Home Economics,
Helwan University.
A. Microbiological study: Confirmation of the probiotics bacteria that
found in the fermented milk (Rayeb) by identification the bacterial
strains was carried out by using specific media and the total counts of
probiotic bacteria were determined.
1. MRS broth medium: MRS broth medium was used to cultivation and
isolation of all species of Lactobacillus in Fermented milk (Rayeb)
according to (De Man et al., 1960). The culture was incubated
anaerobically up to 3 days at 37°C.
2. MRS L-cysteine medium (MRSC): MRS broth, Oxoid supplemented
with L-cysteine hydrochloride 0.05%. (MRSC) broth medium was used
to cultivation and isolation of Bifidobacterium strains (Desjardins et al.,
1990). The culture was incubated anaerobically up to 3 days at 37°C.
Egypt. J. of Appl. Sci., 35 (9) 2020 87
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3. ST agar medium: ST agar medium was suitable for the selective
enumeration of Streptococcus thermophilus from Fermented milk
(Rayeb), (Dave and Shah, 1996).
B. Biological study: The basal diet (g/kg diet ) was consisted of 140 g
casein (> 80 % protein), 100 g sucrose, 40 g corn oil, 50 g cellulose, 35 g
mineral mixtures, 10 g vitamin mixtures, 1.8 g L-cystine, 2.5 g choline
bitartrate and the remainder is corn starch. Diets were formulated
according to (Reeves et al., 1993).
Induction of obesity : Rats were fed on basal diet for four weeks
according to Reeves et al., (1993) with some modification included (high
fat diet) according to Min et al., (2004) containing: casein 14%,
cellulose 5%, vitamin mixtures 1%, mineral mixtures 3.5% , sucrose
10%, ( beef tallow 19% + corn oil 1%), l-cystine 0.18%, choline
bitartrate 0.25 % and the remainder was starch to induce obesity.
After the adaptation period of one week, thirty-five rats were
divided into five groups, (7 rats each). Group (l) was fed on the basal
diet and kept as a control negative (-ve) group. The obese rats (n=28)
were classified into 4 groups, as follows: Group (2): obese rats were fed
on high fat diet only (Control positive group). Group (3): obese rats were
fed on high fat diet and was given orally 1 ml of Rayeb. Group (4):
obese rats were fed on high fat diet and was given orally Vitamin D at
600 IU/Kg BW. Group (5): was fed on basal diet and was given orally a
mixture of (1 ml of Rayeb and Vitamin D at 600 IU/Kg BW).
At the end of the experimental period (8 weeks), rats were fasted
overnight before sacrificing and the blood samples were collected from
each rat then centrifuged for 15 minutes at 3000 r.p.m.to separate serum.
Serum was carefully aspirated and transferred into clean quite fit plastic
tubes and kept frozen at -20 °C until chemical analysis. Peritoneal fat pad
was dissected from the carcasses, then weighed according to the method
of (Azain et al., 2000).
Biological Evaluations: All rats were observed each day. Their feed
(FI) intake was determined daily and body weights were obtained every
week. Body weight gain percent (BWG%) and Feed Efficiency Ratio
(FER) was calculated according to Chapman et al., (1959) using the
following equation:
88 Egypt. J. of Appl. Sci., 35 (9) 2020
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Biochemical Analysis of Serum: Serum total cholesterol (TC)
(Richmond, 1973), triglycerides (TG) (Wahlefeld, 1974), high density
lipoprotein (HDL) (Albers et al., 1983) were determined. Meanwhile,
low density lipoprotein (LDL) and very low-density lipoprotein (LDL)
were calculated according to (Fridewald et al., 1972). Serum Aspartate
amino transferase (AST) and alanine amino transferase (ALT)
(Bergmeyer et al., 1978) were determined. Serum urea (Kaplan, 1984),
uric acid (Patton and Crouch, 1977), and creatinine were measured
according to (Murray, 1984). Leptin hormone was determined using
enzyme-linked immunosorbent (ELISA) assay (Xiong et al., 2005).
Statistical Analysis: The results were expressed as mean ± standard
error (SE). The statistical analysis was carried out by using SPSS, PC
statistical software (Version 20.0 SPSS Inc., Chicago, USA) using the
Dunk 'test multiple range post-hoc test. Data were analyzed by one way
analysis variance (ANOVA). The values were considered significantly
different at (P < 0.05) (Armitage and Berry, 1987).
RESULTS
Regarding to changes in body weight status, Table (1) illustrated
the changes of body weight, feed intake and FER in obese rats fed on
high fat diet supplemented with (Rayeb, VD and their mixture). Rats fed
on high fat diet had significant increase the initial body weight as
compare to the control -ve group, while there were no significant
differences in IBW among all obese groups. The high fat diet
supplemented with (Rayeb, VD and their mixture) significantly (P<0.05)
decreased the FBW compared to the positive control group. There were
significant differences (P<0.05) in FBW among the treated groups. The
lowest decrease in body weight was recorded at the group fed a mixture
of Rayeb and vitamin D.
Table (1). Effect of high fat diet supplemented with Rayeb, Vit D and
their mixture on BW, BWG%, FI and FER of obese rat.
Parameter
Groups
Initial BW Final BW BWG (%) Weight
reduction
%
Feed Intake
(g/day/rat)
FER
(g)
Control (-ve) 150.0±1.87 b 171.50±2.23 cd 14.40±1.91 b - 16.00 0.022±0.028 b
Control (+ve) 201.0±2.06 a 239.41±2.67a 19.18±1.89 a - 21.60 0.029±0.027 ab
G1: Rayeb 199.5±2.51 a 176.50±1.68 c -11.46±1.31cd 26.27 16.70 0.023±0.028 b
G2: Vit D 201.16±1.66 a 187.16±2.61 b -6.93±1.42 c 21.84 17.50 0.013±0.027 c
G3: Mixture 195.83±2.34 a 167.50±1.80 d -14.40±1.45 d 30.03 14.00 0.033±0.037 a
Data are expressed as mean ± SE.
Means with different superscript letters in the column are significantly differences
at (P < 0.05).
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In regarding to BWG%, obese rats (control +ve) had significant
(P<0.05) increased in BWG% as compared to control -ve group. All
treated groups significantly decreased BWG% compared to the positive
control group. The mixture (VD and Rayeb) caused the lowest BWG%
compared to the other treated groups. The percent of weight reduction
was recorded by 26.27%, 21.84%, 30.03% for the groups fed on Rayeb,
Vitamin D or their mixtures, respectively. It was seen that the FI was
lowered for the treated groups compared the control +ve group. FER was
statistical lowered at the groups fed either vitamin D or the mixture (Vit
D and Rayeb) compared to the positive control group.
Results in table (2) illustrated that positive control group had
significant increase (P<0.05) in leptin hormone, compared to the
negative control group, while supplementation with (Rayeb, Vit. D and
their mixture) significantly reduced (P<0.05) the level of leptin
hormone, compared to the positive control group. The highest reduction
in leptin hormone concentrations was recorded at the mixture group. In
regarding to Peritoneal Fat bad, there was significant difference
(P<0.05) in Peritoneal Fat bad concentrations for the treated groups
compared to the +ve control. The greatest decrease in Peritoneal Fat bad
concentrations was obtained by a mixture of Vit. D and Rayeb.
Table (2): Effect of high fat diet supplemented with Rayeb, Vit. D
and their mixture on serum leptin and Peritoneal Fat
bad concentrations of obese rats.
Parameter
Groups
Leptin
(pg/ml)
Peritoneal Fat bad (%)
Control (-ve) 35.75±0.45 e 1.18±0.01 d
Control (+ve) 110.30±1.30 a 6.94±0.37 a
G1: Rayeb 53.00±2.00 c 2.55±0.15 c
G2: Vit D 69.75±1.85 b 3.60±0.10 b
G3: mixture 44.80±0.20 d 2.03±0.03 c
Data are expressed as mean ± SE.
Means with different superscript letters in the column are significantly differences
at (P ≤ 0.05).
Obese rats fed on high fat diet had significant increased (P<0.05)
the level of serum liver functions (AST, ALT and ALP) compared to the
negative control group (Table 3). The treatments with either Rayeb, Vit.
D or their mixture significantly decreased (P<0.05) the elevated levels of
serum AST, ALT and ALP compared to the positive control group. It
was clear that, no significant differences in AST, ALT and ALP levels
among the treated groups. Supplementation with mixture of vitamin D
and Rayeb caused the highest reduction in liver function.
90 Egypt. J. of Appl. Sci., 35 (9) 2020
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Table (3): Effect of high fat diet supplemented with Rayeb, Vit D and
their mixture on liver function of obese rats.
Parameters
Groups
AST ALT ALP
(μ/L)
Control (-ve) 50.04±1.19 c 26.64±1.88 c 1.75±0.09 b
Control (+ve) 74.14±1.90 a 39.76±1.02 a 2.76±0.14 a
G1: Rayeb 63.00±1.81 b 32.76±1.39 b 1.97±0.11 b
G2: Vit D 64.48±1.17 b 34.14±1.13 b 2.08±0.21 b
G3: mixture 62.72±2.31 b 31.00±1.41 b 1.88±0.03 b
Data are expressed as mean ± SE.
Means with different superscript letters in the column are significantly differences
at (P ≤ 0.05).
Table (4) shows the effects of high fat diet supplemented with
(Rayeb, Vit D and their mixture) in serum kidney functions on obese rats.
Feeding with high fat diet significantly increase (P<0.05) the level of uric
acid, creatinine and urea compared to the control –ve. Feeding obese rats
on diet supplemented with (Rayeb, Vit D and their mixture) caused a
significant decrease (P<0.05) in the mean values of uric acid, creatinine
and urea as compared to the positive control group. There were no
significant differences in serum uric acid, creatinine and urea between the
treated groups.
Table (4): Effect of high fat diet supplemented with Rayeb, Vit D and
their mixture on kidney function of obese rats.
Parameters
Groups
Uric acid Creatinine Urea
(mg/dl)
Control (-ve) 1.76±0.15 c 0.66±0.01 c 27.44±1.27 c
Control (+ve) 3.38±0.11 a 1.67±0.09 a 43.74±1.09 a
G1: Rayeb 2.61±0.15 b 1.05±0.08 b 34.44±1.20 b
G2: Vit D 2.51±0.17 b 0.84±0.02 c 32.90±1.24 b
G3: mixture 2.61±0.17 b 0.83±0.02 c 33.48±1.28 b
Data are expressed as mean ± SE.
Means with different superscript letters in the column are significantly differences
at (P ≤ 0.05).
The effect of high fat diet supplemented with (Rayeb, Vit D and
their mixture) on lipids profile of obese rats was illustrated in table (5).
Feeding rats with high fat diet caused a significant increase (P<0.05) in
serum lipid profile, however, serum HDL-C was significantly lowered,
compared to the normal rats. Diet supplemented with (Rayeb, Vit D and
their mixture) significantly decreased (P<0.05) the mean value of serum
TC, TG, VLDL-C and LDL-C, however, serum HDL-C level was
increased significantly (P<0.05), compared to the positive control group.
It was noticed that, no significant difference in TC, TG, HDL,
LDL and VLDL levels among the treated groups. It was obvious that, the
supplementation with Vit D gave the highest beneficial effect in
improving lipid profile in obese rats.
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Table (5): Effect of high fat diet supplemented with Rayeb, Vit D and
their mixture on lipid profile of obese rats
Parameter
Groups
TC TG HDL-C VLDL-C LDL-C
(mg/dl)
Control (-ve) 76.62±1.85c 64.20±1.73c 57.84±0.77 a 12.84±0.34c 5.94±2.04 c
Control (+ve) 108.46±1.26a 87.44±1.62a 39.84±0.60c 17.48±0.32a 51.13±1.87a
G1: Rayeb 92.28±1.48b 71.50±1.19b 50.72±2.15b 14.30±0.23b 27.26±2.39b
G2: Vit D 86.18±2.04b 69.62±2.67b 50.98±2.55b 13.92±0.53b 21.27±2.52b
G3: mixture 88.44±3.05b 71.16±1.07b 48.68±1.23b 14.23±0.21b 25.52±3.43b
Data are expressed as mean ± SE.
Means with different superscript letters in the column are significantly differences
at (P ≤ 0.05).
DISCUSSION
The present research was conducted to evaluate the biological
evaluation of probiotic fermented milk with vitamin D on obese rats. The
probiotics bacteria as bio-starters at the level of "107 CFU/ml" in
fermented milk "Rayeb" including three strains of probiotics
(Lactobacillus acidophilus, Bifidobacterium bifidum, and
Streptococcus thermophilus) were used to examine their beneficial
effect on body weight, leptin hormone, peritoneal, liver, kidney functions
and lipid profile of obese rats.
The normal intestinal microbiota is important for maintaining
host health because it provides energy in the form of short-chain fatty
acids (Cummings and Macfarlane, 1997). Functional dairy products,
specially fermented milk; recently its availability and popularity
increased in the daily-life. Consumers‟ interest about personal health is
reasons in establishing markets for these functional products (Gasmalla
et al., 2017).
Fermented milk (Rayeb) is a popular dairy product with wide
spread worldwide consumption. Studies have reported health benefits of
Fermented milk owing to its probiotic content comprising live Lactic
acid bacteria (LAB) that prevent pathogen growth, by producing organic
acids and bacteriocins in the gastrointestinal tract (Bourlioux et al.,
2003). Additionally, yogurt helps prevent colorectal cancer and diabetes
and helps prevent and manage hyperlipidemia (Cho et al., 2006).
Furthermore, Fermented milk (Rayeb) and yogurt contains various LAB
and stimulates the immune system and stabilizes the gut microflora
(Parvez et al., 2006).
Commonly used probiotics include Lactobacillus,
Bifidobacterium, Bacillus, Saccharomyces, Streptococcus, Clostridium
92 Egypt. J. of Appl. Sci., 35 (9) 2020
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(Cox et al., 2015). More than three kinds of probiotics and prebiotics can
play a role in fighting obesity by reducing intestinal bacterial
lipopolysaccharides, altering bacterial composition, and reducing fat
storage (Swami and Shah, 2017). Probiotics have significant effects on
oxidative stress, inflammatory factors, and lipids with no adverse events
reported. Mechanisms for these obesity related effects include regulation
of immune differentiation and insulin sensitivity, inhibition of pathogenic
bacteria adhesion to the intestine and translocation to adipose tissue, and
improvement of intestinal barrier function. Moreover, treatment of
obesity with a probiotic-rich diet is a major advancement in the treatment
of obesity (DiBaise et al., 2008).
Supplementation with VD to obese rats significantly decreased
the higher body weight, this result is in accordance with some studies
reported by Caan et al., (2007) demonstrated that postmenopausal
women who received vitamin D and calcium supplementation had a
lower weight gain compared to placebo. Ortega et al., (2008) found that
women with higher vitamin D responded more positively to low calorie
diets and lost more body fat during the experimental period.
In addition, Zhu et al., (2013) also supplemented patients with
vitamin D and calcium in a low calorie diet and found that the body
weight reduction was significantly higher than control group.
Vimaleswaran et al., (2013) studied the causality and the relationship
between BMI and VD in a very large number of individuals (up to
42,000 participants) and concluded that obesity leads to a low status of
this vitamin but it is unlikely that the deficiency predisposes to a higher
BMI.
Treatment with VD (2.5-10 mcg/kg/day) produced significant
dose-dependent decrease (p<0.05) in body weight parameters, feed
intake, weight and size of fat pads, levels of serum glucose, TGs, TC, and
LDL as compared to HFD group. Moreover, the level of serum HDLs
was increased as compared to HFD group. So that, VD treatment
ameliorated established obesity and associated biochemical consequences
(Verma et al., 2016). Merino et al., (2017) demonstrate that the
nutrition of rats with diet deficient in VD and high fat, promotes
overweight by increasing fat deposits, suggestion a cause-effect
relationship between VD deficiency and overweight. A significant
amount of VD seemed to be stored in the liver and adipose tissue when
dietary VD is at a supplementation level; thus excess body adiposity
Egypt. J. of Appl. Sci., 35 (9) 2020 93
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could contribute to relatively low serum 25(OH)D level when vitamin D
was supplemented (Park et al., 2020).
Regarding to lipid profile in the current work, obese rats had
lowered (P<0.05) lipid profile due to Rayeb and/or VD supplementation.
Rayeb helps prevent colorectal cancer and diabetes and helps prevent and
manage hyperlipidemia (Cho et al., 2006). Probiotics can inhibit
cholesterol uptake and increase cholesterol efflux by activating liver X
receptors, eventually reducing the overall cholesterol level and potential
risk of developing atherosclerosis (Huang et al., 2015). Parhamfar et
al., (2018) suggests that consumption of milk fermented by L. fermentum
improves serum lipid trends in rats by lowering serum total cholesterol,
triglycerides, LDL-C levels, as well as by increasing HDL-C level. It also
plays a role in the prevention of obesity induced by a high-fat diet.
Moreover, the administration of probiotic-fermented milk
significantly lowered the levels of TC and TG in rat serum and viscera (P
< 0.05) and significantly increased the level of total bile acid in the rat
liver and small intestine (P < 0.05) due to the adhesion and absorption of
living bacteria cells, the production of short chain fatty acids, the
reduction of the reabsorption of bile acids, and the inhibition of
lipoprotein lipase activity (Wa et al., 2019). Additionally, significant
going down of lipid profile, but scale-up of HDL-c were noticed after
dietary supplement with probiotic bacteria in hypercholesterolemic rats.
So, it is useful of fermented milk for improving the prevention of
cardiovascular disease (Allbban, 2020). These results are in accordance
with the obtained results.
Some studies investigating the association between VD levels and
dyslipidemia in obese children have suggested that VD deficiency might
be linked to metabolic syndrome (Rusconi et al., 2015). The functions of
vitamin D are linked to lipid values. VD regulates calcium metabolism
and increases intestinal calcium absorption, thereby reducing intestinal
fatty acid absorption (Wang et al., 2016). Therefore, a reduction in
intestinal fat absorption can lower the cholesterol level. Additionally,
increasing the calcium concentration promotes the conversion of
cholesterol into bile acids in the liver, resulting in reduced cholesterol
level (Vaskonen et al., 2007).
VD level seems to affect the lipid profile, even in non-obese
children, and a low VD level may progress to dyslipidemia or obesity in
non-obese children (Kim and Jeong, 2019). Pashova-Stoyanova et al.,
(2019) confirmed the protective role of vitamin D and its effects in
94 Egypt. J. of Appl. Sci., 35 (9) 2020
11
lowering the elevated uric acid levels. Vitamin D decreases glucose and
uric acid concentrations and improves the cardiogenic lipid profile
(cholesterol/HDL and LDL/HDL). Cho et al., (2020) indicate that the
consumption of yogurt fermented by Lactobacillus fermentum TSI for 8
weeks by obese rats reduces abdominal fat and improves blood lipid
metabolism in HD-induced obese rats.
The current results revealed that supplementation with Rayeb
and/or VD significantly decreased (P<0.05) serum leptin hormone and
perirenal fat pads of obese rats. Obesity is associated with an increase in
the size and amount of adipose tissue (Cani et al., 2009). Adipose tissue
secretes a number of signaling molecules commonly referred to as
adipocytokines (Koerner et al., 2005). Leptin and adiponectin are the
important adipocytokines produced by adipocytes.
In addition, the weights of the mesenteric fat pads, perirenal fat
pads, and epididymal fat pads were lower in the Lactobacillus gasseri
group compared to the control group (Kang et al., 2010)
The obtained results are in agreement with Sundari et al., (2020)
who reported that oral administration of VD (2400 IU) decreased proinflammatory
substances, such as leptin and mRNA MCP-1 and
increased anti-inflammatory substances, such as adiponectin, in visceral
adipose tissue of obese female Wistar rats. The result, regarding serum
leptin, are similar to the other study that reported by (Belenchia et al.,
2013), which state that the administration of high dose VD 2000 IU in
twice daily on obese adolescents rats for 3 months has reduced the level
of leptin significantly, compared to other obese groups which do not
receive the treatment. There is a positive association between leptin level
and body fat mass, it means more body fat more leptin (Erturk et al.,
2004).
The rats fed the Lactobacillus gasseri diet displayed greater numbers of
small adipocytes from mesenteric retroperitoneal adipose tissues and
leptin concentrations were decreased to 32 % than did those on the
control diet. These results indicate a possible role for a fermented milk
product in the regulation of adipose tissue growth (Sato et al., 2008).
The obtained results showed that the liver functions were
significantly improved as results of Rayeb and/or supplementation.
Patients with elevated ALT were found to have lower vitamin D.
(Liangpunsakul and Chalasani, 2011). Low serum levels of vitamin D
have been observed in chronic liver diseases, especially with liver
cirrhosis (Kitson and Roberts, 2012). The number of large-size adipose
Egypt. J. of Appl. Sci., 35 (9) 2020 95
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tissue, the level of triglyceride, leptin and AST were significantly
reduced in obese rats fed on yogurt fermented by Lactobacillus
plantarum (Park et al., 2016). In animal models of liver and
cardiovascular metabolic damage, the supplementation of 23 IU/day/rat
vitamin D3 shows liver and cardio-protective effects (Mazzone et al.,
2018).
Finally, it could be concluded that, consumption of Rayeb,
vitamin D or their mixture might be useful in weight reduction and
alleviate their complications.
REFERENCES:
Albers, N.; V. Benderson and G. Warnick (1983): Enzymatic
determination of high density lipoprotein cholesterol, Selected
Methods, Clin. Chem., 10 :91-99.
Allbban, Amal M. (2020): Influence of Probiotics Supplementation on
the Lipid Profile of Hypercholesterolemic Rats. Journal of Arts,
Literature, Humanities and social sciences, 54.
https://doi.org/10.33193/JALHSS.54.2020.145
Armitage, G.Y. and W.G. Berry (1987): Statistical methods 7th Ed.
Ames., Iowa State University. Press .39-63.
Azain, M.J.; D.B.Hausman; M.B.Sisk; W.P. Flatt and D.E. Jewell
(2000): Dietary conjugated linoleic acid reduces rat adipose
tissue cell size rather than cell number. J. Nutr., 130:1548-1554.
Belenchia, A.M.; A.K.Tosh; L.S. Hillman and C.A. Peterson (2013):
Insufficiency Improves Insulin Sensitivity In Obese
Adolescents: A Randomized Controlled Trial. The American
Journal of Clinical Nutrition.;97(4):774–781.
Bergmeyer, H.U.; P. Schreiber and A.W. Wahlefeld (1978):
Optimization of methods for aspartate and alanine
aminotransferase. Clin Chem., 24:58-61.
Borgeraas, H. ; K. Johnson ; J. Skattebu ; K. Hertel and J.
Hjelmesæth (2018): Effects of probiotics on body weight, body
mass index, fat mass and fat percentage in subjects with
overweight or obesity: a systematic review and meta-analysis of
randomized controlled trials , Obesity Reviews., 19: 219–232.
Bourlioux, P.; B. Koletzko ; F. Guarner and V. Braesco (2003): The
intestine and its microflora are partners for the protection of the
host: Report in Danone symposium the intelligent intestine. Am.
J. Clin. Nutr., 78:675–683.
96 Egypt. J. of Appl. Sci., 35 (9) 2020
13
Caan, B.; M.Neuhouser; A.Aragaki; C.B. Lewis; R.Jackson; M.S.
LeBoff and et al., (2007): Calcium plus vitamin D
supplementation and the risk of postmenopausal weight gain.
Arch Intern Med.;167: 893–902.
Cani, P.; E.Lecourt; E. Dewulf and et al., (2009): Gut microbiota
fermentation of prebiotics increases satietogenic and incretin gut
peptide production with consequences for appetite sensation and
glucose response after a meal. Am J Clin Nutr; 90(5): 1236–
1243.
Chapman, D.; R. Gastilla and J. Campbell (1959): Evaluation of
protein in foods: 1- A Method for the determination of protein
efficiency ratio. Can. J. Biochem. Phys , 37: 679- 86
Chatelier, E.; T. Nielsen; J. Qin; E. Prifti; F. Hildebrand and G.
Falony (2013): Richness of human gut microbiome correlates
with metabolic markers. Nature; 500(7464):541-6.
Cho, W.; G.Hong; H.Lee; S.Yeon; H.Paik; Y. Hosaka and C. Lee
(2020): Effect of Yogurt Fermented by Lactobacillus
Fermentum TSI and L. Fermentum S2 Derived from a
Mongolian Traditional Dairy Product on Rats with High-Fat-
Diet-Induced Obesity . Foods, 9(594):1-12.
Cho, Y.H.; H.J.Shin; C.H. Chang and M.S. Nam (2006): Studies on
the development of the yogurt decreasing blood glucose. Korean
J. Food Sci. Anim. Resour., 26:257–262.
Cotillard, A.; S.Kennedy; L.Kong and et al. (2013): Dietary
intervention impact on gut microbial gene richness. Nature;
500(7464): 585–588.
Cox, C.M.; R.A. Dalloul ; B.S. Jenie ; E. Andreas and A. Yuneni
(2015): Immunomodulatory Role of Probiotics in Poultry and
Potential in Ovo Application. Beneficial Microbes., 6(1):45-52.
Cummings, J.H. and G.T. Macfarlane (1997): Role of intestinal
bacteria in nutrient metabolism. J Parenter Enteral Nutr ., 21:
357–365.
Dave, R.I. and N.P. Shah (1996): Evaluation of media for selective
enumeration of Streptococcus thermophilus.J Dairy Sci.,79 (9):
1529-1536.
De Man, J.C. ; M. Rogosa, and S.M. Elisabeth (1960): A medium for
the cultivation of lactobacilli. Appl. Bact., 23: 130-135.
De Vrese, M. and J. Schrezenmeir (2008): Probiotics, prebiotics, and
synbiotics., Adv Biochem Eng Biotechnol.;111:1-66
Egypt. J. of Appl. Sci., 35 (9) 2020 97
14
Desjardins, M.L. ; D. Roy and J. Goulet (1990): Growth of
Bifidobacteria and their enzyme profiles. Journal of Dairy
Science., 73: 299-307.
DiBaise, J.K.; H. Zhang ; M.D. Crowell ; R. Krajmalnik-Brown ;
G.A. Decker and B.E. Rittmann (2008): Gut microbiota and
its possible relationship with obesity. Mayo Clin. Proc., 83:460–
469.
Erturk, E.; N.Kuru; V.Savci; E.Tuncel; C. Ersoy and S. Imamoglu
(2004): Serum Leptin Levels Correlate With Obesity Parameters
But Not With Hyperinsulinsm In Women With Polycystic
Ovary Syndrome. Fertil Steril.; 82: 1364-1368.
Food and Agriculture Organization. (2006): Probiotics in Food:
Health and Nutritional Properties and Guidelines for Evaluation;
FAO: Rome, Italy,.
Fridewald, W.T.; R.I. Leve and D.S. Fredrickson (1972): Estimation
of the concentration of low density lipoprotein separated by
three different methods. Clin. Chem., 18: 499-502.
Gasmalla, M.A.; H.A.Tessema; A. Salaheldin ; K. Alahmad and H.A.
Hassanin (2017). Health benefits of milk and functional dairy
products. MOJ Food process. Technol., 4(4): 99-108.
Huang, Z.; C.Mu; Y.Chen; Z.Zhu; C.Chen; L. Lan and et al., (2015):
Effects of dietary probiotic supplementation on LXRalpha and
CYP7alpha1 gene expression, liver enzyme activities and fat
metabolism in ducks. Br. Poult. Sci., 56:218–224
Kang, J. H.; S. I. Yun and H.O. Park (2010): Effects of Lactobacillus
gasseri BNR17 on body weight and adipose tissue mass in dietinduced
overweight rats. J. Microbiol., 48: 712- 714.
Kaplan, L.A. (1984): Clin Chem. The C.V. Mosby co.st Louis.Toronto.
Princeton; 1032-1036.
Khayatzadeh-Mahani, A. ; A. Ruckert and R. Labonté (2018):
Obesity prevention: Co-framing for intersectoral „buy-in‟. Crit.
Public Health, 28:4–11.
Kim, R. and S. Jeong (2019): Relationship between Vitamin D Level and
Lipid Profile in Non-Obese Children. Metabolites, 9(125):2-8.
Kitson, M.T. and S.K. Roberts (2012): D-livering the message: the
importance of vitamin D status in chronic liver disease. J
Hepatol., 57:897–909.
Koerner, A. ; J. Kratzsch and W. Kiess (2005): Adipocytokines: leptin,
the classical; resistin, the controversial; adiponectin, the
98 Egypt. J. of Appl. Sci., 35 (9) 2020
15
promising; and more to come, Best Pract. Res. Clin. Endocrinol.
Metab., 19:525-546.
Liangpunsakul, S. and N. Chalasani (2011): Serum vitamin D
concentrations and unexplained elevation in ALT among US
adults. Dig Dis Sci., 56:2124–2129.
Mazzone, G.; C.Morisco; V.Lembo; G.D’Argenio; M.D’Armiento
and et al., (2018): Dietary supplementation of vitamin D
prevents the development of western diet-induced metabolic,
hepatic and cardiovascular abnormalities in rats. United
European Gastroenterol J., 6(7): 1056–1064.
Merino, O.; B.Gregório; F. Sampaio ; R. Sánchez and J. Risopatrón
(2017): Role of Vitamin D in the Development of Obesity. Int.
J. Morphol., 35(4):1568-1575.
Min, L.; S. Ling; L.Yin ; C. Stephen ; J. Randy ; D. David and T.
Patrick (2004): Obesity induced by a high fat diet down regulates
apolipoprotein A-IV gene expression in rat hypothalamus., Am J
PhysiolEndocrinolMetab., 287: E366- E370.
Morelli, L. and L. Capurso (2012): FAO/WHO guidelines on
probiotics: 10 years later. J. Clin. Gastroenterol., 46: S1–S2.
Murray R. (1984): ClinChem the C. V. mosby co. st Louis. Toronto.
Princeton., 1088- 1090.
NCDRFC, (2016):. Trends in adult body-mass index in 200 countries
from 1975 to 2014: a pooled analysis of 1698 population-based
measurement studies with 19.2 million participants. Lancet;
387(10026): 1377–1396.
Ng, M.; T. Fleming and M.Robinson (2014): Global, regional and
national prevalence of overweight and obesity in children and
adults 1980–2013: a systematic analysis. Lancet (London,
England) ; 384(9945): 766–781
Ortega, R.M.; A.Aparicio; E.Rodríguez-Rodríguez; L. Bermejo and
et al., (2008): Preliminary data about the influence of vitamin D
status on the loss of body fat in Young overweight/obese woman
following two types of hypocaloric diet. Br J Nutr.;100(2):269-
72.
Parhamfar, M.; M. Khaleghi ; S. Fahimirad and M. Godarzi
(2018): A study on the Effects of Lactobacillus fermantumfermented
Milk Consumption on Lipid Patterns of Rats Fed with
a High Fat Diet. Journal of Ilam University of Medical
Sciences., 26(1):23-33
Egypt. J. of Appl. Sci., 35 (9) 2020 99
16
Park, C.; Y.Shin; J.Kim; S.Zhu; Y.Jung and S.Han (2020): Effects
of high fat diet-induced obesity on vitamin D metabolism and
tissue distribution in vitamin D deficient or supplemented mice.
Nutrition & Metabolism., 17(44):1068
Park, S.; K. Seong and S. Lim (2016): Anti-obesity Effect of Yogurt
Fermented by Lactobacillus plantarum Q180 in Diet-induced
Obese Rats. Korean J. Food Sci. An., 36(1): 77-83.
Parvez, S.; K.A.Malik; S.A. Kang and H.Y. Kim (2006): Probiotics
and their fermented food products are beneficial for health. J.
Appl. Microbiol., 100:1171–1185.
Pashova-Stoyanova., L.; A. Tolekova ; M.Ganeva ; T.Zhivka; P.
Hadzhibozheva ; T. Georgiev and K. Nancheva (2019):
vitamin d effects on lipid profile and uric acid levels in the
experimental model of metabolic disorders in fructose fed wistar
rats. Farmacia, 67(6):1071-1076.
Patton, C.J. and S.R. Crouch (1977): Calorimetric determination of
blood urea. Analyt. Chem., 49: 464-469.
Pereira-Santos, M.; P.R.Costa ; A.M. Assis ; C.A. Santos and D.B.
Santos (2015): Obesity and vitamin D deficiency: A systematic
review and meta-analysis. Obes. Rev., 16: 341–349.
Perna, S. (2019): Is Vitamin D Supplementation Useful for Weight Loss
Programs? A Systematic Review and Meta-Analysis of
Randomized Controlled Trials. Medicina (Kaunas). 12;55(7).
Reeves, P.G.; F.H. Nielsen and G.C. Fahey (1993): AIN-93 purified
diets for laboratory rodents: final report of the American
Institute of Nutrition ad hoc writing committee on the
reformulation of the AIN-76A rodent diet. J Nutr.,123:1939–51
Richmond, N. (1973): Colorimetric determination of total cholesterol
and high density lipoprotein cholesterol (HDL-c). Clin. Chem.,
19: 1350-1356.
Rusconi, R.E.; V.De Cosmi ; G. Gianluca ; C. Giavoli and C.
Agostoni (2015): Vitamin D insufficiency in obese children and
relation with lipid profile. Int. J. Food Sci. Nutr., 66:132–134.
Sanders, M.E. (2008): Probiotics: definition, sources, selection, and
uses. Clin Infect Dis. 1;46 Suppl 2:S58-61.
Sanders, M.E. (2016): Probiotics and microbiota composition. BMC
Med; 14(1): 82.
100 Egypt. J. of Appl. Sci., 35 (9) 2020
17
Sato, M.; K.Uzu; T.Yoshida and E. Hamad (2008): Effects of milk
fermented by Lactobacillus gasseri SBT2055 on adipocyte size
in rats. British Journal of Nutrition., 99(5):1013-7
Sundari, L.; M.Bakta; N.Astawa; P.Adiatmika; G.Arijana and
K.Tunas (2020): The effect of vitamin D administration on leptin,
adiponectin and mRNA MCP-1 levels in adipose tissue of obese
female wistar rats. Curr Res Nutr Food Sci; 8(2):541-549.
Swami, O. and N.J. Shah (2017): Role of Probiotics in Diabetes: A
Review of Their Rationale and Efficacy.Diabetes.,5:104-110
Vaskonen, T.; E. Mervaala ; V. Sumuvuori ; T. Seppänen-Laakso
and H. Karppanen (2007): Effects of calcium and plant sterols
on serum lipids in obese Zucker rats on a low-fat diet. Br. J.
Nutr., 87: 239–245.
Verma, A.; A.Goyal ; R.Kaur and A. Kamboj (2016): Beneficial
effect of vitamin D on high-fat diet-induced obesity in wistar
rats. Asian J Pharm Clin Res, 9(4): 337-340.
Vimaleswaran, K.; D.J. Berry; C. Lu; E. Tikkanen; S. Pilz; L.T.
Hiraki; J.D. Cooper; Z. Dastani; R. Li; D.K. Houston and et
al. (2013): Causal relationship between obesity and vitamin D
status: Bi-directional Mendelian randomization analysis of
multiple cohorts. PLoS Med.;10:e1001383.
Wa, Y.; B.Yin; Y.He; W.Xi; Y.Huang; C.Wang; F.Guo and R. Gu
(2019): Effects of Single Probiotic- and Combined Probiotic-
Fermented Milk on Lipid Metabolism in Hyperlipidemic Rats.
Food Microbiology, 10:1312.
Wahlefeld, A.W. (1974): Methods of Enzymatic Analysis. Academic
Press, Chapter, 5: 1831-1835.
Wang, Y.; S. Si; J. Liu ; Z. Wang ; H. Jia ; K. Feng ; L. Sun and S.J.
Song (2016): The associations of serum lipids with vitamin D
status. PLoS ONE, 11(10): e0165157.
Watanabe, M.; K. Morimoto and S. Houten (2012): Bile acid binding
resin improves metabolic control through the induction of
energy expenditure. PLoS One; 7(8): e38286.
World Health Organization. (2000): Obesity: Preventing and
Managing the Global Epidemic;World Health Organization:
Geneva, Switzerland.
Xiong, Y.; L. Shen ; K.J. Liu ; P. Tso ; Y. Xiong ; G. Wang; S.C.
Woods and K. Liu (2005): Anti-obesity and antihyperglycemic
effects of ginsenoside Rb1in rats. Diabetes., 59: 2505-2512.
Egypt. J. of Appl. Sci., 35 (9) 2020 101
18
Zhu, W.; D.Cai; Y. Wang; N.Lin; Q.Hu; Y.Qi; S.Ma and S.
Amarasekara (2013): Calcium plus vitamin D3
supplementation facilitated fat loss in overweight and obese
college students with very-low calcium consumption: A
randomized controlled trial. Nutr J.;12:8. doi: 10.1186/1475-
2891-12-8.
التقییم البیولوجی للَبن المخمر"ال ا ریب" المحتوی علی بادئات حیویة وفیتامین د
على الفئ ا رن المصابة بالسمنة
أمنیة جلال رفعت ، محمد احمد عرفو، نعیم محمد ا ربح ، رنیم صلاح الدین صبره
لقد تم استکشاف البروبیوتیک عمى نطاق واسع فی التصنیع والإنتاج الغذائی والبحث
العممی والوقایة من الأم ا رض ، لذلک أجریت هذه الد ا رسة لمتقییم البیولوجی لمَبن المخمر"ال ا ریب"
المحتوی عمی بادئات حیویة وفیتامین د عمى الفئ ا رن المصابة بالسمنة لمدة 8 أسابیع. صُنفت
الفئ ا رن )ن = 53 ( إلى 3 مجموعات عمى النحو التالی: المجموعة ) 1(: تتغذى عمى النظام
الغذائی الأساسی وتبقى کمجموعة ضابطة سالبة. بینما تم تغذیة الفئ ا رن )ن = 88 ( عمى نظام
غذائی عالی الدهون لمدة أربعة أسابیع لاحداث السمنة ثم قسمت إلى أربع مجموعات ،
المجموعة ) 8( تم تغذیتها عمى نظام غذائی عالی الدهون فقط وکانت بمثابة مجموعة ضابطة
موجبة. المجموعتان ) 5 و 4(: تم تغذیة الفئ ا رن البدینة عمى نظام غذائی عالی الدهون وتم
إعطاؤها عن طریق الفم 1 مل من المبن ال ا ریب وفیتامین د بمعدل 066 وحدة دولیة / کجم من
وزن الجسم عمى التوالی. المجموعة ) 3(: تم تغذیتها عمى الغذاء الاساسى وتم إعطاؤها عن
طریق الفم خمیط من ) 1 مل من الحمیب ال ا ریب وفیتامین د عند 066 وحدة دولیة / کجم وزن
الجسم( .أشارت النتائج إلى أن تناول المبن ال ا ریب وفیتامین د أو خمیطهما أدى إلى انخفاض
٪ فی وزن الجسم النهائی مقارنة بمجموعة الکنترول الموجبة بنسبة 80.82 (P < معنوی ( 0.05
و 81.84 ٪ و 56.65 ٪ عمى التوالی نتیجة انخفاض تناول الغذاء و نسبة کفاءة الغذاء فی
الفئ ا رن البدینة. علاوة عمى ذلک ، انخفض ترکیز هرمون المبتین وکذلک نسبة الدهون البریتونیة
لجمیع المجموعات المعالجة. لوحظ أن التدعیم بالالبان المخمرة (P < بشکل معنوی ( 0.05
وفیتامین د أدى إلى تحسین وظائف الکبد والکمى بالإضافة إلى صورة الدهون فی الفئ ا رن البدینة.
یمکن القول بان تناول المبن ال ا ریب وفیتامین د أو خمیطهما قد یکون مفیدًا فی إنقاص الوزن.
102 Egypt. J. of Appl. Sci., 35 (9) 2020
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