Human intestinal tract contained a diverse number of microbial communities which performed a significant role in human health. The presence of gut microbiota was affected mainly by diet. Camel milk is the source of nutrition and provides all the essential nutrients for growth. It has great significance in the treatment of liver, spleen, and anemic infections. Camel urine has also many medical advantages. In this study we examined the effect of camel milk and urine and a mixture of both (milk + urine) on the growth of Gut microbiota using an in vivo animal model. Fresh fecal samples were collected before and after administration of the tested materials. After that, the microbial analysis was conducted via culturing, denaturing gradient gel electrophoresis and metabolic analysis via high-performance liquid chromatography (HPLC). The result indicated that the numbers of bacterial groups were increased after the first dose. Coliform group have significant increase when given a mix of milk and urine compared to control group with P < 0.05. Bifidobacterium group have significant increase in their number in the Milk and Mix groups compared to control group with P < 0.05. The concentration of Short-chain fatty acids in fecal samples was increased in Milk and Mix groups compared to control group. In conclusion, drinking camal milk, urine or a mix of both increased the growth of Gut microbiota.
The human gastrointestinal tract (GIT) extends from the oral cavity to the rectum and is colonized by a large number of microorganisms. There are more than 1014 organisms in the human colon alone with at least 400 species, approximately 10 times more than the sheer number of somatic cells [
For many decades, the natural products were applied in the fields of medicine, pharmacy, biology and have very essential role in health care and prevention of diseases [
Fifteen albino wistar male rats of the same age with a body weight of 200 - 250 g were collected from the Animal House Unite of King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia. All the studies were conducted in accordance with the ethics and regulation of local governing authorities. Animals were housed in respective plastic cages and divided into three groups: Milk, Mix and control groups. Each group has 5 animals. All experiments were run in the same controlled environment (21˚C ± 2˚C, 65% relative humidity and 12: 12 hour light/dark cycle).
Camel milk and urine were collected from camels that graze in south Jeddah, Saudi Arabia from adult female in the morning in sterilize bottles and kept at 4˚C until used. A daily dose of camel milk (1.8 ml/200 g) was given to the first rat group and a dose of 2.4 ml/200 g of mix (camel milk + urine, v/v) was given to the rat second group through oral gavages for one week. The third group is a control that drank water instead of milk or urine.
Fresh fecal samples were collected at different time points in sterile plastic containers. Containers were kept under anaerobic conditions in anaerobic Jar. Each week, three fecal samples/week were collected from each rat for two weeks, labeled, and then stored at −20◦C until used.
The bacterial groups that were tested, the selective media and culture conditions that were used were shown in
Short chain fatty acids (SCFAs) like Acetic, Propionic, Isobutyric and Lactic were investigated in fecal samples of the three tested animal groups. Water ex- tract from fecal samples were prepared [
The selective media | Bacteria | Incubation Temperature | Incubation Period | Incubation Condition |
---|---|---|---|---|
MRS agar | Lactobacilli | 37˚C | 7 days | Anaerobic |
Beerns agar | Bifidobacterium | 37˚C | 7 days | Anaerobic |
Azide blood agar | Streptococci | 45˚C | 3 days | Aerobic |
MacConkey agar | Coliform | 37˚C | 1 day | Aerobic |
Bacterial DNA was extracted from 200 mg of frozen fecal samples (−20˚C) using QIAamp DNA stool Mini kit, according to the manufacturer’s instructions. The DNA concentration was measured using Nanodrop spectrophotometer and quality was evaluated using gel electrophoresis. The DNA that isolated from collected fecal samples was used as a template in PCR amplification. The V3 region of 16S rRNA was targeted to amplify the total DNA bacterial community using universal primer. The forward primer (341GC-F) (5’CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG G CCT ACG GGA GGC AGC AG 3͗ ) GC-clamp is in boldface) and the reverse primer (534R)(5͗-ATT ACC GCG GCT GCT GG-3͗) were used [
In this study, the DNA that was extracted from fecal samples from all experimental groups of rats at different time points were analyzed using DGGE which was performed using Bio-Rad Protein 11 system, essentially as described by [
To investigate the histological changes in the rat colons, sigmoid colon sections, stained using Hematoxylin and Eosin and examined using light microscope [
Statistical analysis was applied using the statistical Package software for Social Science (SPSS for windows, version 16). The significance of the difference between groups was determined using the One-Way ANOVA. The difference was regarded significant when P < 0.05 and non-significant when P > 0.05.
After administracion of camel milk and mix (camel milk and urine) to rats for a week, seven rat fecal samples were collected during 2 weeks. Each fecal sample was cultivated on the selective media to investigate the different groups of bacteria in the rat feces. The data revealed that using camel milk or a mix of both milk and urine increased all types of tested bacteria compared to control (zero time). Results of the enumeration of cultivable bacteria are presented in
The results also indicated that the Coliform group has the highest growth in the Mix fed group with 1.6 × 109 cfu/ml, after the first week. This increase was significant compared to control group with P < 0.05. The growth of the same group of bacteria was also increased in Milk fed group (2.2 × 105 cfu/ml) compared to control. The enumeration of Streptococci bacteria showed the highest growth in the Mix fed group with 1.3 × 105 cfu/ml, while 1.7 × 104 cfu/ml in the Milk feed group. However this increase was not significant in comparison to control group (P > 0.05). The results also showed that the Lactobacilli group was increased in numbers to 2.4 × 106 cfu/ml and 2.3 × 106 cfu/ml in Milk and Mix groups, respectively. Bifidobacterium group showed similar increase in their growth with 1.42 × 106 and 1.46 × 106 cfu/ml in the Milk feed group and Mix fed group, respectively. This increase was significant compared to the control group with P < 0.05. In the milk fed group, the growth of Coliform and Streptococci
were decreased after stopping the milk doses while the decrease was not clear in case of Lactobacilli and Bifidobacterium. In the mix fed group, the growth of all bacterial groups decreased after the last dose.
In the HPLC chromatograms that were obtained from fecal water analysis, different concentrations of SCFAs were obtained. The result of the average concentrations of the SCFAs is presented in
individual rat. The bands in each line represent different bacterial group. The intensity of the band may provide an assessment of the fraction of the target bacterial group in the sample. The microbial diversity was evaluated by the count of bands existence in the DGGE patterns.
The milk feed group showed a stable number of bands through the dosing period. The mean of number of bands was 8 ± 1 bands. When comparing the intensity of bands in this group, most bands had an increase in their intensity after administrated of milk. The bands with Rf = 0.009, 0.026 and 0.849 were increased by about 2 - 3 folds and these increases were significant (P = 0.001). One band with Rf = 0.196 has imperceptibly increase. The increase in the intensity of those bands indicates an increase in the numbers of these bacterial groups.
Interestingly, in the group that was feed on mixture of milk and urine, the bacterial diversity remained the same after the first three doses while after the fourth dose there was a substantial increase in microbial diversity (number of bands 9 ± 1) with P= 0.003. Additionally, although there were no increase at the first three time points, the intensity of one of the bands (Rf = 0.101) has ten folds increase after the third feeding point. This increase was translated into a rise in number of this particular group of bacteria. On the other hand there was a significant decrease in the intensity of another band (Rf = 0.331) which indicates the loss of numbers of this bacterial group (
Microscopic examination of the colon tissue showed that there are no changes in the structure of the colon tissues (colonic mucosa, submucosa and muscular), but there was an increase in the intensity of the mucus layer in the treaded groups compared to control group, as indicated in
Microbiota in gastrointestinal tract plays a key role in human health [
The enumeration results of fecal bacteria which investigated before and after camel products administrations indicated that the numbers of tested bacterial groups were increased after the first doses. This might be due to the presence of Lactic acid bacteria (LAB) in camel milk, while some researchers reported that lactic acid bacteria (LAB) exist in numerous food products, also are part of natural microbial community in gastrointestinal tract [
Intensity of bands | Rf value | Rat group | |
---|---|---|---|
After dosing (after 2 weeks | Before dosing (zero time) | ||
8392.78 | 4970.17 | 0.009 | Camel Milk |
12153.93 | 4204.19 | 0.026 | |
53157.14 | 22322.36 | 0.849 | |
89460.79 | 81779.19 | 0.196 | |
142938.88 | 14008.48 | 0.101 | Mix (camel milk + urine) |
11281.34 | 32068.27 | 0.331 |
stated that the camel milk represent a source of biological material in dairy products, due to the existence of lactic acid bacteria which are beneficial bacteria. Moreover, more free amino acids and peptides are found in camel milk than in bovine milk which is digested by microorganisms, and therefore, camel milk presents a higher metabolic activity when used in a culture preparation [
Most previous studies have monitored the shift in DGGE patterns by correlating the presence and absence of bands, or switch in the intensity of a single band on the same gel [
The main products of gut bacterial fermentation in the colon are the short- chain fatty acids (SCFAs) which can be detected in fecal samples. However, the actual proportion of these products depends on many factors such as diet [
In this study, sigmoid colon was selected to examine the histological changes in the colon using light microscope, where its main objective is to store feces until enters the rectum and expelled through the anus, and it is the site of wide range of complications. Ulcerative colitis and Crohn’s (inflammatory bowel diseases) may occur here. Also, the diverticulitis is more common in the sigmoid than any other part of the bowel, as well as cancers that prefer the sigmoid colon. The mucus layer functions as a dynamic protective barrier and interacts with the commensal microbiota to keep a steady maintenance and balance. This homeostasis between mucus and the microbiota is damaged in a variety of intestinal disorders, including IBD, where as in these patients, mucosal inflammation was shown to be associated with a reduction in the diversity of the microbiota. More specifically, a loss of anaerobic bacteria such as Bacteroides, Eubacterium, and Lactobacillus species was reported [
The results obtained in this study suggested that the presence of free amino acids and Lactic acid bacteria in Camel milk may play an important role as a natural source to improve the microbial community in the gastrointestinal tract. These results may encourage the consumption of camel milk as a source of nutrition providing the essential nutrients for growth and development of our metabolic activity.
The authors acknowledge with thanks Prof. Faten khorshid who provided us with the pure PMF fraction and helped with the dosing calculation, the study design and project supervision.
Noor, S.O. and Alenini, M.S. (2018) Effects of Oral Administration of Camel Milk and Urine on Gut Microbiota: Biochemical and Microbiological Profiling in Rats. American Journal of Molecular Biology, 8, 1-12. https://doi.org/10.4236/ajmb.2018.81001