Prebiotic potential and other health benets of prebiotic mixture of pigeon pea (Cajanus cajan L) in Wistar NIN rats

The legume prebiotics, especially galacto-oligosaccharides, are the center of health since they play a crucial role in maintaining gut health. The aim of the present study was to understand the role of prebiotic galacto-oligosaccharides of pigeon pea (Cajanus cajan L) on gut microbiota, obesity and type 2 diabetes mellitus in Wistar National Institute of Nutrition NIN rats. Proximate composition and oligosaccharide content of the pigeon pea was estimated and for animal experiment, four-week-old weaning male Wistar National Institute of Nutrition NIN rats (N = 36) were divided equally into three groups (n = 12) control, ranose, and pigeon pea prebiotics groups. All the groups were fed with the respective diet for 18 weeks. After 18 weeks 5 ml of blood was drawn from the retro-orbital plexus and the rats were sacriced by (CO2) inhalation. Among the studied groups, the pigeon pea prebiotic supplementation signicantly (p (cid:0) 0.05) lowered body weight gain (284.7 ± 36.4 grams) and resulted in lower body mass index (3.79 ± 0.18kg / m 2 ). The fat percent was signicantly lower in pigeon pea prebiotic fed group (7.8 ± 2.7) than ranose (11.6 ± 3.1) and control (13.8 ± 2) groups. Pigeon pea prebiotic feeding signicantly (< 0.05) lowered blood glucose levels; it also improved the Homeostasis model assessment of insulin resistance (HOMA IR) index in pigeon pea prebiotic group (32.08 ± 5.37). Pigeon pea prebiotic feeding signicantly (p (cid:0) 0.05) enhanced the fold increase of lactobacillus and bidobacterial count when compared to ranose fed group. The present study explores the role of pigeon pea prebiotics on gut microbiota and the regulation of some of the features of metabolic syndrome such as obesity and diabetes.


Introduction
The biochemical disorders like type 2 diabetes mellitus (T2DM) and cardiovascular diseases (CVD) are considered as signi cant social issues, re ecting the rising morbidity levels in developing and developed nations. The past years have witnessed paradigm shifting alterations in the lifestyle and consumption of macronutrients, which led to an increased rampancy of metabolic irregularities. T2DM patients possess an elevated endangerment of cardiovascular disease, the leading reason for death [1]. Diabetes mellitus is a chronic ailment that contracts when the pancreas does not produce ample insulin, or when the produced insulin cannot be used by the body effectively [2].
As per the International Diabetes Federations data, about 415 million people were affected with diabetes mellitus (DM) in 2015, and this gure may be anticipated to rise up to 642 million by 2040. Indians, about 69.1 million patients suffering from diabetes, are evaluated to be the 2nd largest gure of cases globally following China, in 2015. According to the studies, more than 90% of T2DM are overweight or obese [3]. In India, obesity is amongst the major predisposing conditions for the onset of diabetes and has emerged as an essential public health problem. Other common health consequences of obesity are CVDs, musculoskeletal disorders, and some cancers.
Probiotics and prebiotics are evidenced to be useful supplements for reducing some of the symptoms of metabolic disorders. As per the International Scienti c Association for Probiotics and Prebiotics (ISAPP), the scope and de nition of prebiotics has been changed as "a substrate that is selectively utilized by host microorganisms conferring a health bene t. This de nition expands the concept of prebiotics, possibly include non-carbohydrate substances" [4]. Their action is based mainly on a modulation of the intestinal micro ora. Various research has reported that probiotics and prebiotics engage in the relieving of T2DM and CVDs [5]. Prebiotics are indigestible carbohydrates and bene cial to the hosts [6,7]. Bene cial in uences of the alteration of intestinal micro ora are, the formation of shortchain fatty acids (SCFA), culminating in a lowering of the perception of satiety, fat cells, and insulin resistance [8,9]. The intestinal micro ora has a vital part in developing adipose tissues and managing the irregularities of energy homeostasis [10]. HOMA IR (Homeostasis Model Assessment Insulin Resistance) is a test for insulin resistance in T2DM patients. Many studies reported the correlation between HOMA IR and hyperinsulinemia.
Non-digestible carbohydrates like Galacto-oligosaccharides (GOS) and Fructo-oligosaccharides (FOS) have already emerged as prebiotics. The recent studies on ra nose have also demonstrated prebiotic properties and possess health bene ts. These days' legume prebiotics, especially galactooligosaccharides, are the center of health since they play a crucial role in maintaining gut health [11].
These remain undigested in the upper gastrointestinal tract because of the absence of α-galactosidase enzyme but gets fermented in the colon and results in by-products, particularly short-chain fatty acid known as butyrate. Butyrate executes a necessary part in reducing in ammation, increasing nutrition incorporation, and modulating the intestine [12,13].
Non-digestible carbohydrates play a role in stimulating the incorporation of various minerals and improving the mineralization of bones. Therefore, they are likely additives for "functional foods or nutraceuticals." Besides nutrition-related effects, nutraceuticals have psychologic and physiological advantages that elicit enhanced well-being and lowered risks of chronic ailments. Often, much of the scienti c evidence for the practical implications of non-digestible carbohydrates is on animal studies where in the elevated the obtain ability of calcium, magnesium, zinc, and iron [14].
Data from human experiments and animal models support the advantageous effects of prebiotic on energy homeostasis, management of satiety, and body mass. Dietary intake of prebiotics has been shown a heightening of calcium (Ca) absorption, bone calcium accretion, and bone mineral density [15].
Prebiotics and inulin type fructans are already proven to enhance bones' mineralization and increase calcium and iron absorption. Some studies in animal models had shown high calcium absorption when the diets supplemented with inulin and oligofructose [16].
Low intake of Ca causes conditions such as hypertension, osteoporosis, incidences of human cancer, and renal lithiasis. lactose, prebiotics and such carbohydrate moieties have been reported to be bene cial regulators of Ca retention in the intestine [17]. Based on their componential galacto-oligosaccharides, legumes seem to be explored as nutraceuticals or functional foods, especially with regards to their prebiotic prospective [18]. In India, legumes such as pigeon pea are largely eaten as dhal for protein due to its economically viable nature. The role of pigeon pea prebiotics in health may include advantages on chronic liabilities like immune function, colonic health, weight management, and cardiovascular diseases.
Data regarding the prebiotic potential of pigeon pea oligosaccharides is sparse.
Our earlier study has aided us in understanding the bene cial impact of pigeon pea prebiotics in hypercholesterolemia and weight gain management. Pigeon pea prebiotic fed groups showed improved lipid metabolism by decreasing serum triglycerides, total cholesterol, LDL cholesterol, and VLDL cholesterol in comparison with the control group. Hence, prebiotics can be a preventive measure for obesity and type 2 DM [19]. Therefore, the present study shows the effects of pigeon pea prebiotic oligosaccharide potential on gut microbiota, obesity, and type 2 DM in WNIN rats.

Legume samples
Cultivated pigeon pea samples available in local markets were procured from twin Cities of Hyderabad and Secunderabad, Telangana State. The study is inaccordance with relevant guidelines for plant study Geographically, Hyderabad is located at 17.366°N Latitude and 78.476°E Longitude. Dust and foreign particles were removed from the seed samples by passing them through a screen, and then stored at 4 0 C for further studies. Standards such as verbascose, stachyose, and ra nose used in the experiment were from Sigma Chemicals Co. (St. Louis MO, USA). Only analytical gradedchemicals were utilized for this study.

Nutrient analysis
Proximate composition, moisture, ash, crude ber, and fat components were analyzed by the AOAC, 2006 methods 934.01, 942.05, 962.09, and 920.39, respectively. AOAC Kjeldahl method (984.13) was employed for the analysis of protein(N X 6.25); and the modi ed anthrone technique for carbohydrate estimation [20].

Extraction and evaluation of oligosaccharides
Various pigeon pea seed varieties were grinded to powder and ltered using a 250mm sieve. 5g of the powdered specimen was put into an Erlenmeyer ask (250ml volume) carrying 50 ml 70% ethanol (v/v) and propped onto an orbital shaker for 12 h at 140 rpm. The constituent residue was washed with 25 ml 70% ethanol after its contents were passed via a Whatman No.1 lter paper for ltration. Ethanol from the combined ltrates is evaporated at a temperature of 40°C using a rotary vacuum evaporator. The residue remaining after the vacuum evaporation was dissolved in 10 ml distilled water to obtain concentrated sugar syrup. Then, highperformance liquid chromatography (HPLC) with refractive index (RI) detector was utilized to estimate the oligosaccharide content in it [21].

Animals and Diet
The study was initiated after the ethical approval from the Institutional Animal Ethical Committee (IAEC: P22/IAEC/NN/2012/7/Rat-WNIN-Male-72), National Institute of Nutrition (NIN), Hyderabad, India. All the methods were performed at NCLAS in accordance with the guidelines and regulations of the Committee for the Purpose of Control and Supervision of Experimentation on Animals. (CPCSEA, India). The study is reported in accordance with ARRIVE guidelines. Four-week-old weaning male WNIN rats (n = 36), with an average weight of 63.16 ±1.18 g (mean ± SD), were used for the study. The animals were housed individually in sterilized wire mesh bottomed polypropylene cages and maintained under standard lighting conditions (12-hour light/ dark cycle). Temperature and relative humidity were kept constant at 22±2 0 C and 50±5% respectively. The animals were attuned for a week (7 days) in solitary rates with the basal diet. Three groups of rats, consisting of 12 rats each were supplemented with the experimental diets for 18 weeks. After 18 weeks, the wholly palpable consequences of the test diets will instigate, affecting lipid accumulation inmesentery, adipocyte hyperplasia, elevated fat ratio, and DM2. A basal diet consisting of 250g casein, 50g corn oil, 10g AIN-93G vitamin mixture, 35g of the American Institute of Nutrition Rodent Diets (AIN-93G) mineral combination, 2.5g choline bitartrate, 50g crystalline cellulose, and 602.5g of sucrose per kg of the feed preparation was given to the control group. Table 2 depicts the dietary constituents of the experimental and control diets.
A standard diet augmented with 30g/kg ra nose was given to the ra nose group (RAF) rats. The control animal group was given the standard feed augmented by 30g/kg of pigeon pea prebiotics. Food intake and body mass were documented weekly. Succeeding 18 weeks of the test supplements, 5 ml blood was obtained out of the retro-orbital plexus of the animals. Drawing blood via the retro-orbital plexus is an excellent non-terminal option when in need of an aseptic sample. The rats are then sacri ced by inhalation of carbon dioxide (CO2). Owing to its availability, safety, and immediate onset of action, carbon dioxide (CO2) is customarily used as a euthanasia medium for experimental lab animals. Inhaling huge dose of CO 2 elicits a swift lowering of the intracellular pH, instantly emanating in a lowered performance of the central nervous system (CNS) and death.

Dietary consumption and gain in body weight
Dietary consumption was recorded daily for each group during the experimental period. Feed e ciency and gain in body weight were calculated using the formula "The Body mass index (BMI) is interpreted as the body weight (kg) divided by the square of the body height (m) and is expressed in units of kg/m 2 ."

Body Composition
After the experiment's end, the body composition of the experimental rats was carried out by Total Body Electrical Conductivity (TOBEC) (EM SCAN/ TOBEC, Model SA-3000 Multi-detector, Spring eld, III, USA).
This apparatus estimates the electrical conductivity of the total body of smaller organisms in a noninvasive manner. In control and experimental animals, the estimation was carried out using a coil with I.D. 3076. The experimental rats were made stable and then surveilled for body composition, according to the direction laid out for the apparatus [22,23], and the body composition variables were acquired using mathematical derivations [24].
Bone mineral density (BMD) and bone mineral concentration (BMC) were determined by making use of Dual X-ray absorptiometry (DEXA) (Hologic Discovery QDR 1000 series, cross by drive, Bedford, MA, USA). This instrument measures bone minerals and body composition of smaller species in a non-invasive manner. The examination of the outcome was followed by using imaging software (QDR) given from the manufacturing company (Hologic ultra-high-resolution short animal software version 2.1) in order to analyze the region-wise lipid accumulation in the control and experimental rats.
Oral Glucose Tolerance Test (OGTT) WNIN male rats were kept in fasting for 12 hours (overnight) before the glucose tolerance test. The test was performed by One Touch (J&J) Glucometer. The blood sample was drawn from the tail region of the rat. During the fasting period, the animals were allowed free access to drinking water. 20% glucose stock solution was prepared by dissolving 2 g of D-glucose, in 10 ml of distilled water. The glucose solution was sterilized by passing it through a 0.2µm syringe lter. Before performing a glucose tolerance test, body weight, and a baseline glucose level were recorded for each rat. The injection timing was noted, and additional blood specimen was drawn at 30, 60, 90, and 120 minute intervals to calculate post-challenge blood-sugar levels. GCGTGCTTAACACATGCAAGTC; Reverse Primer: CACCCGTTTCCAGGAGCTATT). The ampli cation conditions are as follows: initial denaturation was carried out at 94ºC for 2 min, followed by thirty cycles of denaturation (10 s,95ºC), annealing (30 s, 60ºC) and extension (30 s, 72ºC) and one nal extension (10 min, 72ºC) for all the primer sets leading to amplicons over 100 bp. The relative fold gene expression was calculated by the 2 -∆∆Ct formula by Kenneth J. Livak and Thomas D. Schmittgen.

Statistical analysis
The nal outcomes were conferred as the average from 3 reproductions along with standard deviation (SD). The average outcomes were analyzed for the extant of deviation using a one-way analysis of variance (ANOVA). The result was evaluated by making use of the Statistical Package of the Social Sciences (SPSS) 20 form.

Results
Proximate composition and oligosaccharide content of pigeon pea seeds The outcomes of the proximate and oligosaccharide component analysis are given in Table 1. The protein content in pigeon pea seed was found to be 25.46%, fat 1.65%, ash 3.66%, available carbohydrates 54.23%, moisture 8.50%, and total bre 6.50%. The sucrose component was determined as 2.44%, ra nose 1.26%, stachyose 1.72%, and verbascose 4.80%.

Food intake and gain in body weight
The diet consumption was calculated based on the average food consumption of all the animals in a group over time (Figure 1a). The average food consumption of pigeon pea prebiotic fed group showed higher (36.51 ± 4.4) when compared to food intake of control (23.31±1.8), and ra nose fed group (24.76 ± 2.4). The difference in the food intake between the control animals and ra nose standard group was almost negligible, whereas pigeon pea prebiotic fed group has shown signi cantly higher food intake (p 0.05). Feed e ciency proportion was remarkably (p 0.05) higher in the control group (3.5± 1.5) when compared to ra nose (3.13 ± 0.7) and pigeon pea prebiotic (1.98±0.5) fed groups (Figure 1b).
The body weight of the animals was recorded weekly throughout the study period, as shown in (Figure  1c). The gain in body weight of ra nose fed animals (381± 43.4) was considerably high (p 0.05) in comparison with the control (346 ± 41.7) and pigeon pea prebiotic fed group (284.7± 36.4).
The body mass index (BMI) results are reported in Figure 1d. From the gure, it is clear that the ra nose supplemented group has shown higher BMI (4.28 ± 0.23 kg/m 2 ) when compared to control (4.01± 0.26 kg/m 2 ) and pigeon pea prebiotic group (3.79± 0.18 kg/m 2 ). Pigeon pea prebiotics supplementation not only elicited in a lesser gain in body mass but also lowered the BMI.

Body Composition
Body composition results are shown in Table 3. There was a remarkable (p 0.05) difference in lean mass, total fat, and fat percentage. The fat percent was shown signi cantly lower in pigeon pea prebiotic fed group (7.8 ± 2.7) than ra nose (11.6±3.1) and control (13.8 ± 2) groups ( Figure 2). Fat-free mass (FFM) was low in pigeon pea prebiotic group (156± 10) compared to ra nose (175.5± 7.2) and control (163.6± 5.9) animals. The three experimental groups showed considerable distinction in the bone mineral content (BMC). BMC content was high in ra nose (303± 4.2) and pigeon pea prebiotic fed group (285 ±18.5) than that of the control group (230±45), whereas no notable distinction of BMD was found among the groups.
Oral glucose tolerance test (OGTT) The outcomes for the OGTT are represented in Table 4 and Figure 3. The table shows that the higher blood glucose level in ra nose than that of control and pigeon pea prebiotic fed groups. The animals fed with pigeon pea prebiotics have signi cantly (p<0.05) showed lower blood glucose levels for all the time intervals compared to ra nose and control groups. It has been studied that the non-digestible carbohydrates alter postprandial glycemia and insulinaemia in rats. The effect of galactooligosaccharides diet upon glucose sensitivity in WNIN rats was studied. Animals that were fed with the GOS augmented food displayed higher glucose utilization in comparison to the rats supplemented alongside a rodent feed alone.
The results of the homeostasis model assessment of insulin resistance (HOMA IR) are demonstrated in Figure 4A. The fasting blood sugarconcentrations were seen to be extremely low in pigeon pea fed group (3.64±0.44 mmol/L) compared to ra nose fed (4.62 ± 0.46 mmol/L) and control group (5.54±0.35 mmol/L). Fasting insulin levels were signi cantly low in pigeon pea fed groups in contrast to the ra nose standard fed group (p 0.05) ( Figure 4B). HOMA -IR score was signi cantly high in the ra nose fed group (47.93 ± 3.24) when compared to control (30.79 ±1.64) and pigeon pea fed group (32.08 ± 5.37). The results depict that fasting insulin levels were in correlation with the HOMA IR score. The HOMA -βeta cell function scores were also signi cantly high for ra nose standard fed group (1000.34 ± 101.29) followed by pigeon pea fed (727.11 ± 143.79) and control diet-fed groups (501.43 ± 45.73) respectively ( Figure 4 C and D).

Quanti cation of DNA
The results of the gut microbiota were given in Figure 5. Bi dobacteria and lactobacilli are the major strains which play a vital role in maintaining gut health hence we demonstrated the effect of pigeon pea supplementation on fold increase of these two strains when compared with control and ra nose fed diets. The gure shows that the fold increase of lactobacillus and bi dobacteriain in pigeon pea galactooligosaccharide fed group is signi cantly high at (P<0.05) in contrast to the reference standard ra nose supplemented animals.

Discussion
Pigeon pea is a vital reserve of proteins, minerals, and vitamins in a plant-based dietary regimen; it also acts as an excellent supply of carbohydrates and dietary bre. Raja et al. [25] has carried out HPLC to nd out the RFOs and sucrose proportion in thirty-two varieties of pigeon pea cultivars. Many human involved researches have manifested that the intake of de nite prebiotic supplements will remarkably change the use of intestinal micro ora. In an earlier study, we have reported the hypolipidemic action of pigeon pea (Cajanus cajan L.) prebiotic oligosaccharides in WNIN rats [19]. A research was organized to assess the outcome of 3 differing prebiotics, i.e., indigestible dextrin, α-cyclodextrin, and dextran on human gut micro ora. The correspondence between prebiotics and intestinal micro ora modi es the decreased pH and in turn, emanated the manufacture of short-chain fatty acids (SCFA) [26]. Studies disclosed the ability of prebiotics in regulating various processes comprising of food assimilation, production of satiety hormones, and modi cations in the intestinal micro ora in a dose-dependent fashion [27]. A study examined discrete and amalgamated effects of the prebiotic oligofructose (OFS) and probiotics on intestinal micro ora and host metabolism in obese mice. Prebiotics signi cantly lowered the weight gain, fat mass, and energy intake [28]. Genetic inclination as well appears to be correspondingly crucial for the onset of obesity, apart from a high-fat diet [29].
Yang et al. [30] have found that the effect of chickpea on visceral fat, dyslipidemia, and insulin resistance in rats. The body fat percent was more in the high fat-fed group animals, and lower in high fat + chickpea fed group. In a study, the experimental animals were supplemented for 2 months with a basal feed, a high fat feed and a high fat feed accompanied by 1-2% freeze-dried black adzuki bean ethanolic extract powder (BAB-1 and BAB-2). The overall body mass and the epidermal fat mass were remarkably lowered. In comparison with the high-fat diet fed animals, the serum lipid pro les of the animals supplemented with the BAB (black adzuki bean) containing diet was enhanced [31]. In a study, when experimental rats fedona high-cholesterol diet were supplemented with encased symbiotic microbes, it exponentially lowered the LDL-cholesterol compared to every one of the other groups, and also highly increased the HDL-cholesterol levels [32].
Okazaki et al. [33,34] have engaged a combination of xylose, xylobiose, xylotriose, and more sugars as a source of carbon for in vitro fermentations with Bi dobacterium infantis, Bi dobacterium longum, and Bi dobacterium adolescentis and have observed that the latter has exhibited a notable capacity to utilize both xylobiose and xylotriose. Hopkins et al. [35] have conducted similar studies using commercial XOS and have determined that the capability of the Bi dobacterium species for propagating on XOS considered strain.
The results suggest that non-digestible GOS plays a role in carbohydrate metabolism [23]. De Cossío et al. [36] have evaluated the effect of persistent provision (2 months) of prebiotics (oligo-fructose) on biophysiological alterations such as glucose homeostasis, body weight, and food intake, characterizing db/db mice. Prebiotic supplementation decreased diet assimilation and blood-sugar related disorders (glucose tolerance and insulin resistance) in db/db mice. In a study, mice were fed with a standard chow feed forti ed with/without inulin from chicory (Fibruline 5%) for 21 days. Glycemic and sucrase activity in the small intestine was lowered after insulin treatment [37]. In another study, lean and obese male JCR: LA-cp rats were given any 1 of the 4 experimental feeds -control, high ber, high protein, or prebiotic ber inulin, for 21 days.OGTT was carried out to analyze glucose, insulin, and plasma GLP-1. Overall, combination of high protein with high fat in the feed elevated GLP-1 production in reaction to oral glucose administration [38]. The gut microbiota is involved in chronic ailments such as type 2 diabetes and obesity. When mice were subjected to prebiotic treatment for eight weeks, it improved Reg3g expression and improved intestinal homeostasis with an increase in the appearance of infection, an essential protein involved in intestinal epithelial cell turnover [39].
In a study, a diet supplemented with Black Adzuki Bean (Vigna angularis) extract with (0.2 mg/mL) dosage signi cantly improved hyperglycemia and HOMA-IR in C57BL/6J obese mice induced by high-fat diet [40]. Hallam and Reimer [41] have studied the effect of high prebiotic ber weaning diet, control on adiposity, and insulin resistance in Wistar rats. When high-fat diet induced diabetic rats were administered with 109 CFU of Bi dobacterium animalis ssp. lactis 420, it appeared to lower the in ammatory cytokines such as IL-1β, PAI-1, TNF-α, and IL-6 and also to improve insulin sensitivity [42].
Dietary prebiotics is interpreted as a speci cally fermented component which supervenes in select alterations in the constitution and/or action of the gastrointestinal micro ora, thereby best owing advantages on host well-being [43]. Davani-Davari et al. [44] have reported that among various compounds that have been studied to assess their function as prebiotics, galacto-oligosaccharides -one of the most common among them, are found in signi cant amounts in chickpea. Fernando et al. [45] have illustrated that the chickpea diet modulated the intestinal micro ora of subjects with potentially bene cial effects associated with a rise in Bi dobacterium sp. and a fall in pathogenic and putrefactive bacteria including Clostridium clusters. Hou et al. [46] demonstrated that high-fat diet formulated with whole green gram or decorticated green gram promoted the growth of Bacteroidetes and reduced the growth Firmicutes, which helped in the restoration of gut health. Xu-Dong Yang et al. [47] have reported that galacto oligosaccharides' administration will induce a major modi cation in the beta heterogeneity of the intestinal micro ora and the multiplication of Bi dobacterium species and other possible antiin ammatory microorganisms. Burokas et al. [48] reported that the synergistic administration of fructooligosaccharides (FOS) and GOS induces a growth in the comparative occurrence of Lactobacillus. Chen et al. [49] have showed that administration with the prebiotic fructooligosaccharides from the medicinal plant Morinda o cinalis can elevate the lactobacillus constitution. This study is the rst to reveal the usage of red gram prebiotics to alter the intestinal microbiota composition, according to my awareness.

Conclusion
The signi cant un abating biochemical discrepancies like insulin resistance and type 2 diabetesleads to higher morbidity and mortality. Varying processes are involved in the relation between gut micro ora and these diseases. Amidst the strategies used to regulate intestinal micro ora, prebiotics and/or probiotics seems like the most advantageous/bene cial, even though the real data is yet restricted. Legumes have been investigated as functional foods based on the galacto-oligosaccharides as prebiotics which modulate the colonic microbiota in humans since it is shown to signi cantly increase levels of bi dobacterium spp. and decrease production of pathogenic bacteria. The current experiment investigates the role of pigeon pea prebiotics on gut microbiota and the regulation of some of the characteristics of the metabolism-related syndromes like obesity and diabetes. The effect of pigeon pea prebiotic supplementation on the fold increase of lactobacillus and bi dobacteria in the red gram prebiotic supplemented animals is signi cant (P < 0.05) upon juxtaposing with ra nose fed animal groups. The red gram prebiotic on body composition and OGTT in Wistar NIN rats was signi cant at 95% con dence interval. Supplementation of diet with pigeon pea prebiotics showed a lower gain in the body weights of WNIN rats.

Declarations
Data availability: All data generated or analysed during this study are included in this published article.   Values are mean ±SD of 6 rats * a,b the mean difference is significant at the (p<0.05) level Values are mean ±SD of 6 rats * a, b the mean difference is significant at the (p<0.05) level.  Total body fat percentage of control and experimental groups. Error bars indicate standard deviation (SD). * The mean difference is signi cant at the (p<0.05) level.

Figure 3
Page 19/20 The Oral glucose tolerance test of control and experimental groups. Error bars indicate standard deviation (SD). * The mean difference is signi cant at the (P<0.05) level.