Efficacy and Enzymatic Synthesis of Prebiotic Galacto-Oligosaccharides from Lactose Against Colon Carcinogenesis in Male Rats

In the present study the role of galacto-oligosaccharides (GOS) was assessed against development of colon cancer in rats. This study was based on the object to synthesize prebiotic galacto-oligosaccharides from lactose and to assess the protective role of galacto-oligosaccharide against colon cancer. Research was carried out in two phases, production of prebiotic galacto-oligosaccharide and assessment of their protective role against colon cancer. Highest %age of enzyme activity was observed at 6.5 pH and 37°C. Transgalactosylation was carried out and the process was carried out at 37°C using three different concentrations of enzyme. Animals belonging to prebiotic galacto-oligosaccharides groups (G2, G3 and G4) and inulin (Go) had significantly decreased aberrant crypt foci (ACF) counts per colon as compared to controlled animals (G1 and G5). The body mass of animals belonging to galacto-oligosaccharides (Group G2, G3 and G4) increased significantly higher as compared to control group (G1). Effect of pre-biotic treatment had significant effects on organ weights; liver, heart, spleen as well as small intestine length. In our findings galacto-oligosaccharides (GOS) were found to be protective against colon cancer however further research is needed at DNA level to find out the mutations at gene levels.


Introduction
Functional foods exert a vital role in the prevention of nutritionrelated diseases. A food said to be functional contains new and technologically advanced ingredients having some specific health benefits [1]. Among most frequently used functional food compounds are probiotics, prebiotics, vitamins, and calcium and plant antioxidants [2]. Colon cancer is the third leading cause of cancer-related deaths in whole world and is fourth most common cancer type. Mortality rate associated with colon cancer is high because of insidious onset of this disease.
Dietary habits can either increase or decrease the risk of colon cancer. In this aspect, much importance is given to probiotics such as Lactobacilli or Bifidobacteria, and prebiotics such as fructans or fructo-oligosaccharides. As, some studies have reported that their intake is associated with less occurrence of chemically induced colon cancers in animal models. Functional foods play a vital role in preventing nutrition-related diseases. A food said to be functional contains new and technologically advanced ingredients having some specific health benefits [1]. Among, most frequently used functional food compounds are probiotics, prebiotics, vitamins, calcium and plant antioxidants [2]. Gut micro flora consists of different populations of microorganisms including harmful as well as health promoting microorganisms. A wide variety of interactions take place between these microorganisms. In order to improve colonic health, minimize the risk of various diseases and well-being of host, it is necessary to achieve a "balanced gut micro flora". Various dietary strategies could be implied to modify composition of colonic microbiota [3]. More recently, prebiotics are being used as a strategy for obtaining balanced microflora. These are non-digestible ingredient which positively affects the host by selective stimulation of activity or growth of one or limited number of colonic bacteria thereby improving host health" [4]. Colon cancer is the third leading reason of cancer-related deaths in whole world and is fourth most common cancer type. Mortality rate associated with colon cancer is high because of insidious onset of this disease. Dietary habits can either increase or decrease the risk of colon cancer. In this aspect, much importance is given to probiotics such as Lactobacilli or Bifidobacteria, and prebiotics such as fructans or fructo-oligosaccharides.
Several studies concluded that galacto-oligosaccharides (GOS) help promote immunity by exhibiting inhibitory effect on enteric pathogenic infections. They cause maximum decrease in harmful Clostridiaascompared to another pre-biotics [5]. Galactooligosaccharides (GOS) exhibit various beneficial effects on human health. Other than maintaining a balanced micro flora, they also enhance digestibility of dairy products and lactose tolerance, reduction in serum cholesterol levels, risk of cancers, enhance mineral absorption [6]. Aim of present research was to find out effect of prebiotic treatment on the onset of colon cancer.

Enzyme production
Escherichia coli (E. coli) containing beta-galactosidase gene from Lactobacillus reuteri L103 was cultured as the source of the βgalactosidase enzyme. E. coli cells were grown on Luria-Bertani broth (LB) containing the appropriate antibiotics (100 μg/mL ampicillin) required for maintaining the plasmids. Bacteria was grown at 37°C in incubator for 14-16 hours in 5 ml LB medium, then 1-5% of this culture was transferred into 40 ml medium and was grown again at 37°C for 14-16 hours. 1-5% of this culture was transferred into 2000 ml medium and was grown again at 37°C until optical density of this media was between 0.3-0.6, then isopropyl-β-D-thiogalactoside (IPTG) was added (final conc. in media 0.1 mM), after adding IPTG, bacteria was grown at 25°C for 16 hours.

Thin layer chromatography (TLC)
After transgalactosylation process the samples were used for thin layer chromatography and for the quantification of galactooligosaccharides. The samples were diluted in the ratio of 1:9 by taking 20 µl of the sample and 180 µl of distilled H 2 O and mixed properly before using for the TLC. 1.0 µl from each sample along with standards (glucose, galactose, lactose) was taken and applied on silica gel plate. Running buffer used in TLC was prepared by water, n-propanol, ethanol, and n-butanol in the ratio of 2:3:3:2 respectively. Silica gel plates were put in the chromatographic tank till the buffer reached the 2/3rd of the plate at the upper end. TLC was run for 45 minutes. Now the plates were taken out of the chromatographic tank and dried in the hot air oven at 100°C. Staining buffer (0.6 g thymol+5 ml H 2 SO 4 (95%) + 95 ml ethanol) was sprayed on the dried plates. Sugar stains were visible after 110°C heating in hot air oven.

Quantification of galacto-oligosaccharides
Quantification of Galacto-oligosaccharides was done by measuring lactose, D-galactose and D-glucose through Megazyme kits (Megazyme International, Ireland).

Calculation of galacto-oligosaccharide
Total Galacto-oligosaccharides produced were calculated by following formula:

Efficacy studies
For efficacy study 54 male Sprague Dawley rats weighing 135-190 g were used. Approval was taken from Ethical Committee of University of Veterinary and Animal Sciences, Lahore. Rats were housed under 12-hour light-dark cycles, 55 ± 5°C relative humidity and temperature 23 ± 2°C (Figure 2). Water and standard pellet diet were given. Induction of colon cancer: Cancer was induced in all rats by using 1, 2-Dimethylhydrazine dihydrochloride (DMH). DMH was prepared in 1 mM EDTA and pH was set at 7.0 using 1 mMNaOH. DMH was injected subcutaneously at a dose of 40 mg/kg body weight twice a week for two weeks [7] (Figures 3 and 4).
Experimental design: After receiving last dose of DMH, rats were randomly divided into six groups, one positive control group (G0), one negative control group (G1), three treatment groups (G2, G3, G4) and one control group (G5). Each group consisted of nine rats and they were kept in separate cages. Before any experimental intervention, all rats were fed on standard basal diet for a period of one week. Negative control group (G1) was fed on standard basal diet alone. Positive control group (G0) was fed on inulin (84 mg/100 g body weight), while treatment groups G2, G3 and G4 received 60 mg, 84 mg and 107.14 mg/100 g body weight respectively of GOS along with standard diet for a period of 60 days. Nasogastric feeding tube was used for administration of GOS and inulin. Three rats from each group were   Organ to body weight ratio: After dissection, organs i.e., liver, kidney, heart, spleen, small intestine, large intestine was collected to evaluate the effect of test diets on different organ weights of rats. Organs were cleaned and weighed on weight balance [8]. Intestine length was measured with a measuring scale. Results were expressed as organ to body weight ratios (g/100 g of body weight).
Liver and kidney function tests (LFTs and RFTs): Serum was analyzed for liver functioning through determination of enzymes; alkaline phosphatase (ALP) and Alanine Aminotransferase (ALT). Kidney function was also evaluated through serum creatinine and urea assessment [9].
Enumeration of aberrant crypt foci (ACF): After decapitation, the colon was removed, and aberrant crypt foci were counted from the distalpart of colon which was cut into 2.5 cm long small sections. Section was stained using 0.2% methylene blue dye and aberrant crypt foci were counted by using light microscope. The total number of aberrant crypt foci/ rat was calculated as the sum of the large, medium and small aberrant crypt foci [10].

Statistical analysis
Results were analyzed by applying one-way analysis of variance (ANOVA) followed by Post Hoc LSD tests and were expressed as mean ± standard deviation(SD). The differences among groups were considered significant when p ≤ 0.05.
Liver enzymes: Alkaline transaminase (ALT) results showed a significant difference for G0 (Inulin+DMH) group with respect to DMH treated group (G1) and all three galacto-oligosaccharide groups G2, G3 and G4. Among prebiotic groups, G2 and G3 came out to be significantly different with respect to G4 and non-significant with respect to each other. For Alkaline phosphatase (ALP), no significant difference was observed between Inulin+DMH group (G0) and any of three galacto-oligosaccharide groups G2, G3 and G4 but a significant difference with respect to G1 (DMH+basal diet). Also, all three galacto-oligosaccharide groups were non-significantly different with respect to each other.

Discussion
Lactulose, fructo-oligosaccharides (FOS), and galactooligosaccharides (GOS) are those prebiotics which currently fulfill the three criteria defined for prebiotics and they clearly increase the number of lactobacilli and bifidobacteria thereby altering the balance of large intestinal microbiota [11]. A positive association between reduction in experimental colon cancer and prebiotics, probiotics or synbiotics (combination of probiotics and prebiotics) has been observed [12]. Thus, in the present study, the protective effects of GOS against chemically induced colon cancer was investigated in male rats. Significantly reduced number of aberrant crypt foci (ACF) and tumors in colon were seen in those groups which were fed on GOS. These results are in concordance with previous studies suggesting that prebiotics have the capability of minimizing the occurrence and variety of colon cancers which are chemically induced [12]. An intervention study was carried out in pre-menopanusal women who were obese as well as dyslipidemia. According to this study, safe and recommended daily ingestion of GOS was suggested to be 14 g [3]. Findings from a placebo crossover study suggest that ingestion of 20 g of GOS daily has a positive effect on accelerating colonic transit time, when fed to healthy individuals [10]. There are numerous studies supporting the fact that GOS show modulating effects on tumor succession both in rat and human colon carcinogenesis. In another study, modulating effects of GOS consumption on cell propagation was investigated. Proliferation of tumor cells can lead to high risk of cancer [13]. During the development of tumors in colon, an increase in apoptosis (cell death) and cell proliferation has been observed. It is also reported that apoptosis inducing effect of synbiotic or prebiotic is more significant shortly after cancer induction as compared to in last phase of colon cancer [14]. There are various studies suggesting that fructooligossacharides or galacto-oligosaccharides are helpful in early biomarkers on colon carcinogenesis [15][16][17].

Conclusion
Animals belonging to prebiotic galactooligosaccharides groups (G2, G3 and G4) and inulin (Go) had significantly decreased ACF counts per colon as compared to controlled animals (G1 and G5). The body mass of animals belonging to galactooligosaccharides (Group G2, G3 and G4) increased significantly higher as compared to control group (G1). Effect of pre-biotic treatment had significant effects on organ weights; liver, heart, spleen as well as small intestine length.