INTRODUCTION
Schistosomiasis is a serious health problem since it affects about 200
millions of the world population in developing countries, while 400-600
millions are exposed to the risk of infection. The importance of the disease
lies in the fact that it affects not only the overall health status and
fitness of the people, but also the human productivity and national economy.
There is thus a need to apply efficient methods for transmission control
in conjunction with chemotherapy and vaccine development. In recent time
the concept isproviding functional foods containing healthful components
rather than removing potentially harmful ones. Functional foods, designer
foods, pharma-foods and nutraceuticals are synonyms for foods with ingredients
that can prevent and treat diseases (WHO, 1995; Madwar et al.,
1983, 1989). Flat worms belong to the genus Schistosoma; includes species
which are parasitic in humans and domestic animals. At least more than
200 million people suffer from schistosomiasis (Bilharzia) in the tropics
and sub-tropics. Infection occurs through direct penetration of the skin
by the water-dwelling infective stage; thereafter the parasite (which
achieve 1-3 cm in length) are blood-dwelling either in the veins around
the bladder or the intestine, depending on the species involved (Atkinson
et al., 1999).
A functional food is similar in appearance to, or may be, a conventional
food, is consumed as part of a usual diet and is demonstrated to have
physiological benefits and/or reduce the risk of chronic disease beyond
basic nutritional functions (Goldberg, 1994).
Labneh is a type of dairy products manufactured from milk and fermentation
was carried out using lactic acid bacteria. A probiotic is a live microbial
feed supplement which beneficially effects on the host animal by improving
its intestinal microbial balance. Hence, it may be considered as a functional
food with the special property of containing live and beneficial microorganisms
(Fuller, 1992; Mclntosh, 1996).
Garlic (Allium sativum) and onion (Allium cepa) are used
all over the world for different diseases (Ibn-cenna, 980-1937 A.P.).
More of than 4000 publications have provided evidence for the efficacy
of these herbs in the prevention and treatment of a variety of diseases
and for validating its traditional uses. It has been shown that garlic
has different applications as antimicrobial (Azad Chowdhury et al.,
1991; Yoshida et al., 1998), antitumor (Karasaki et al.,
2001; Sundaram and Milner, 1996), antithrombotic, hypolipidaemic, antiarthritic
and hypoglycemic agent (Duraka et al., 2002; Kumar et al.,
2003).
The therapeutic use of probiotics has been considered as successful in
the cases of lactose intolerance, certain diarrheal diseases, Inflammatory
Bowel Disease (IBD) and atopic eczema (Fuller, 1992; Mercenier et al.,
2002). Positive indications have been obtained (and which still need to
be reinforced) for Irritable Bowel Syndrome (IBS), colon cancer, travelers
diarrhea and H. pylori infection. In contrast, insufficient proof
has been provided for some effects on viral infections or blood cholesterol
reduction. They are used for modulation of the immune system improving
resistance to chemicals, inflammation and other factors by binding and/or
degradation of potential carcinogens (improved intestinal metabolic activity).
They also used for alteration of the metabolic activities of the intestinal
microflora (production of antitumorigenic and for antimutagenic compounds)
and for improvement (quantitatively/qualitatively) of the intestinal microflora,
reducing the putative producers of carcinogens and cancer promoters (improvement
of the intestinal microecology). This can be achieved by more bile-acid
degrading bacteria; less bacteria producing the enzymes azoreductase,
nitroreductase, betaglucuronidase, beta-glucosidase etc. (Mercenier et
al., 2002).
The aim of the present study is to evaluate the effectiveness of probiotic
labneh containing garlic or onion oil against Schistosoma mansoni
in infected mice.
MATERIALS AND METHODS
Milk Protein Concentrate
ALAPROTM 4700 milk protein concentrate powder was purchased
from NZMP Co., New Zealand. The milk protein concentrate composition was
4.4% moisture, 69.8% total protein, 14% fat, 7.2% ash and 17.20% lactose.
Garlic and Onion Oils Preparation
Garlic and onion oils were extracted and purified from the Egyptian
crops using local pressing and squeezer, Cairo, Egypt.
Traditional and Probiotic Cultures
Starter cultures of Streptococcus salivarius subsp. thermophilus,
Lactobacillus delbrueckii subsp. bulgaricus and DVS-ABT2
(containing Streptococcus thermophilus, Lactobacillus acidophilus
and Bifidobacterium bifidum) were purchased from Chr. Hansen`s
Lab., Copenhagen, Denemark.
Preparation of Traditional and Probiotic Labneh
Milk protein concentrate powder (ALAPROTM, NZMP Co., New
Zealand) was reconstituted and standardized to achieve 16% total solids
contents and then heated at 85°C for 30 min, cooled to 40°C and
divided into two portions:
| • |
The first portion (P1) was inoculated with
DVS-ABT2 starter (containing Streptococcus thermophilus,
Lactobacillus acidophilus and Bifidobacterium bifidum)
and supplemented with 3% garlic oil (manufactured in local pressing
and mangle). |
| • |
The second portion (P2) was inoculated with DVS-ABT2
starter (containing Streptococcus thermophilus, Lactobacillus
acidophilus and Bifidobacterium bifidum) and supplemented
with 3% onion oil (manufactured in local pressing and mangle). |
All portions were incubated for 4-8 h at 42°C. After coagulation,
the curd was pH tested and stored at 4-6°C±2.
Diets
The compositions of basal diets used in this study are milk protein
(12%), sucrose (5%), fat (10%), vitamin mixtures (1%), mineral mixtures
(4%), dietary fiber (4%) and starch (64%).
Animals
Thirty female Swiss albino mice (18-25 g) were randomly divided into
three-test groups (each containing 10 mice). Experimental animals were
placed in cages and given the basal diets only or basal diet supplemented
with probiotic labneh containing garlic and onion oil for 21 before infection
and then, 45 days after infection:
| • |
The first group (G1) animals were fed on
the basal diet plus probiotic labneh containing 1% garlic oil (5 g/mouse/day). |
| • |
The second group (G2) animals were fed on the basal diet
plus probiotic labneh containing 1% onion oil (5 g/mouse/day). |
| • |
The third group (G3) animals served as control and were
fed on basal diet. |
Infection and Experimental Groups
After twenty one days post feeding mice were infected subcutaneously
(S.C.) with 100 Schistosoma mansoni cercariae per each
mouse.
Assessment of Worms Burden
Perfusion and recovery of adult worms were performed at 6 weeks post
infection through hepatic portal vein by the perfusion method
(Smithers and Terry, 1965). The total tissue worms in liver and intestine
were determined. Protection was assessed as the percentage reduction in
worm counts in liver and intestine according to the formula:
Where:
| P |
= |
Percentage reduction of worms |
| C |
= |
Mean worm burdens in control infected animals |
| T |
= |
Mean worm burdens in pre-treated infected animals |
The percentage reduction in ova counts in liver and intestine can were
also, calculated according to the previous equation.
Mean and standard Error of the obtained data from each different experimental
group were calculated and analyzed according to the method described by
Miller and Miller (1992).
Methods of Analysis
Chemical composition and sensory evaluation for the appearance, color,
flavor and overall properties of synbiotic fermented milk were adopted
from NASA (1999) and analyzed according to official method (AOAC, 1990).
Sensory evaluation were carried out using twenty panelists with three
categories : (+++) very good (++) good, (+) accepted, (-) unaccepted.
RESULTS AND DISCUSSION
The food industry is the largest industry in the world aiming to increase
in the development of foods that promote health. It includes foods such
as natural functional foods (Honey, herbs, fermented milk and cereal,
cranberry juice, green tea etc.), foods and ingredients for specified
health use, formulas (infant and elderly), medical foods, nutraceuticals
and drug foods. Within this continuum between food and drug, there are
unlimited niches for the development of food systems that promote optimal
health and general well-being. Probiotic and herb have a therapeutic effect
that are responsible for enhancing human health and nutrition (Fuller,
1992).
Chemical composition and nutritional values of probiotic labneh (g/100
g) are shown in Table 1.
Sensory evaluation of probiotic labneh containing garlic and onion oil
are presented in Table 2. The results indicated that
labneh with garlic or onion oil rated lower in the sensory evaluation
than plain labneh but that they were still good and acceptable in all
categories.
The effect of probiotic labneh containing garlic and onion oil have demonstrated
an anti-schistosomal activity against Schistosoma mansoni-infected
mice. The obtained results showed that probiotic labneh containing garlic
and onion oil have a protective effect against worm burden of S. mansoni
in infected mice compared with control (Table 3). Moreover,
data showed that the levels of protection against schistosomiasis were
66.67, 57.14 and 50.0% reduction in total worm couple, male and female
in mice infected with S. mansoni fed on probiotic labneh containing
garlic oil. Also, the levels of protection against schistosomiasis in
mice infected with S. mansoni fed on probiotic labneh containing
onion oil were 50, 57.14 and 50% reduction in total worm couple, male
and female, respectively (Table 4).
| Table 1: |
Chemical composition and notional values of probiotic
labneh (g 100 g) |
 |
| Table 2: |
Sensory evaluation properties of synbiotic fermented
milk |
 |
| +++: Very good, ++: Good, +: Accepted, -: Un-accepted |
| Table 3: |
Effect of probiotic labneh containing garlic and onion
oil on worm burden of S. mansoni in infected mice |
 |
On the other hand, probiotic labneh containing garlic and onion oil reduced
ova count in liver and intestine of Schistosoma mansoni infected
mice compared with infected mice fed on basal diet (control). In this
respect probiotic labneh containing garlic oil are more efficient than
probiotic labneh containing onion oil (Table 5).
The results exhibit that mean values of total ova count in intestine
of infected mice fed on probiotic labneh containing garlic and onion were
613.40±2.75 and 626.87±1.61, respectively, compared with
ova count of infected mice fed on basal diet (2723.0±1.37). However,
the corresponding values of total ova count in liver of were 625.70±1.59
and 947.2±0.50 for garlic and onion oil, respectively, compared
with ova count of infected mice fed on basal diet (2174.6±3.45).
These values result in reductions in total ova count in liver and intestine
ranging between 56.44-76.98% and 76.98-77.48 in infected mice fed on probiotic
labneh containing onion and garlic oil (Table 6). Moreover,
garlic oil was more effective in the liver, but the reduction was almost
similar for both oil in the intestinal total ova count.
The mean percentage of reduction in total ova count in liver were 70.0
and 56.44% in mice infected with S. mansoni and fed on probiotic
labneh containing garlic and onion, respectively. The mean percentage
of reduction in total ova count in intestine of mice infected with S.
mansoni and fed on probiotic labneh containing garlic and onion were
77.48 and 76.98%, respectively (Table 6). The mean values
of immature, mature and dead oogram in mice infected with S. mansoni
and fed on probiotic labneh containing garlic oil were 44.0±1.47,
43.0±3.5 and 10.0±2.5, respectively and 47.0±2.82,
46.0±0.77 and 6.0±1.57 in mice infected with S. mansoni
and fed on probiotic labneh containing garlic oil, respectively compared
with mice fed on basal diet (39±1.89, 53.0±2.03 and 5±1.89)
(Table 7). Probiotic labneh containing garlic oil slightly
have more fetal effect on mean values of dead oogram than onion oil in
mice infected with S. mansoni.
| Table 4: |
Percentage of worm reduction of probiotic labneh containing
garlic and onion oil of S. mansoni infected mice |
 |
| Table 5: |
Effect of probiotic labneh containing garlic and
onion oil on ova count in liver and Intestine of S. mansoni
infected mice |
 |
| Table 6: |
Percentage of ova reduction of probiotic labneh containing
garlic and onion oil in liver and Intestine of S. mansoni infected
mice |
 |
| Table 7: |
Effect of probiotic labneh containing garlic and onion
oil on number of immature, mature and dead Oogram of S. mansoni-infected
mice |
 |
The obtained results showed that probiotic labneh containing garlic and
onion oil have been demonstrated an anti-schistosomal activity against
Schistosoma mansoni infected mice. In this, study, probiotic strains
of lactic acid bacteria such as Streptococcus thermophilus,
Lactobacillus acidophilus and Bifidobacterium bifidum were
used to prepare the probiotic labneh. Probiotic strains have a beneficially
affects in human health and used to correct the traditional nutrient deficiencies
by improving its intestinal balance (Fuller, 1992). Rey et al.
(1983) used milk with oltipraz in the urinary schistosomiasis patients.
They used a single dose of 35 mg kg-1 with a drug used to treat
schistosomiasis (oltipraz), which was given under surveillance together
with either whole milk or herrings in oil. The tolerance of the product
was very good as only 3% of the patients reported vomiting and 3% paresthesias
of the fingers. Sixty six percent (66%) of the patients examined on day
30 and/or on day 90 were egg-negative and egg excretion was reduced by
at least 90% in 22 other subjects, bringing the percentage of good results
to 74%. On day 90, the mean egg excretion was reduced by 82.5% in the
overall population and by 80.9% in the 5 to 14 year-old.
The obtained data are in agreement with data previously reported from
large number of epidemiological studies which assessed the influence of
milk intake on infectious diseases developed it showed that the major
milk proteins, casein, whey proteins and membrane structures, may all
exert stimulative effect on the immune system and preventive effect of
many of diseases such as cancer and infectious diseases. Whey proteins
were found to be protective elative to other protein sources (McIntosh,
1993), this being associated with an increase in the intracellular level
of glutathione (GSH), where whey is a prime source of precursors. When
liver glutathione levels rise, the liver is able to more effectively detoxification.
Un-denatured whey protein optimizes serum and liver glutathione levels
(McIntosh et al., 1995).
Also, our result are in agreement with finding reported with Maghraby
et al. (2005) who investigated the anti-schistosomal activity of
colostral and mature camel milk on Schistosoma mansoni infected
mice. They found that colostral and mature milk of camel have an immunoprotective
response against Schistosoma mansoni. The study suggested that
camel milk can be used with antischistosomal drugs in Schistosomiasis
patients. Finding this study are in agreement with the finding of Ghanem
et al. (2005) who studied the immunmoprophylactic effect of yoghurt-containing
probiotic feeding before and after infection with Schistosoma mansoni.
They reported that supplementation of yoghurt containing probiotic decreased
the enlarged spleen and liver weights, almost to the control level to
be nearest the control. Yoghurt-containing probiotic has immunomodulatory
effect and anti-schistosomal activity against Schistosoma mansoni.
Another study have demonstrated that parasite engulfment and destruction
by macrophages is significantly enhanced in Balb/c mice
treated with either garlic extract or its immunomodulator fraction (Ghazanfari
et al., 2005).
Garlic enhances protective immunity against Leishmaniasis is an infectious
disease infection by various mechanisms. Garlic has been shown to activate
nitric oxide synthase (NOS), the enzyme that produces NO, in platelet
and placental villous tissue (Das et al., 1996). On the other hand,
activated macrophages produce NO as an important effector molecule and
it has been confirmed that control of Leishmania infection in he
murine model is NO-dependent. Macrophages from resistant strains of mice
expressed significantly higher levels of NOS and produced larger amounts
of NO compared to the susceptible strains when activated in vivo
(Mcsorley et al., 1996). Based on this information and the results
from this study, they suggest that intra macrophage parasite destruction
with garlic may be due to NOS activation as an important effector mechanism
in macrophage.
Garlic possesses the ability to inhibit the growth of parasites in the
intestines. Garlic has also been used in folk medicine in many parts of
the world to treat pinworms, an annoying but generally harmless intestinal
parasite. Also, Studies were continued on the pathogenesis of systemic
lupus erythematosus, antioxidant effects of garlic/onion oil, development
of novel uterine relaxants, pharmacokinetics of rifampicin and ofloxacin
and role of angiotensin converting enzyme inhibitors in myocardial stunning
ICMR-NIC (2001). Another study was carried out by Kalantari and Salehi
(2001) which reported that Garlic oil, as similar to N-acetylcysteine,
can eliminate electrophilic intermediates and free radicals through conjugation
and reduction reactions. Therefore it protects the liver from toxic doses
of acetaminophen. They also observed the protection by the garlic oil.
The clearance of the toxic metabolites of the acetaminophen from the liver
occurs much faster in immediate treatment with garlic oil (200 mg kg-1).
Results of the present study revealed that probiotic labneh containing
garlic and onion oil demonstrated a protective effect and reduced the
total worm couple, male and female as well as, ova in liver and intestine
in mice infected with S. mansoni. Data, also, showed that probiotic
labneh containing garlic or onion oil increased the dead oogram in mice
infected with S. mansoni. It can be concluded that probiotic labneh
containing garlic and onion oil may be play a great role as a protective
food against infectious diseases such as Schistosomiasis. Furthermore,
the garlic oil surpassed the onion oil on their protective effects. Further
studies are required to investigate the biochemical and immunological
consequences in the humans given such these oils.
ACKNOWLEDGMENTS
The Authors thank Prof. Dr. Hassan M. Mousa and Prof. Dr. Mustafa M.
Zayton, College of Agriculture and Veterinary Medicine, Qassim University
for their reviewing and scientific advise and for reviewing the manuscript.
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