Fecal Metabolomes in Response to Feed Supplemented with Fermented Parkia biglobosa and Sphenostylis stenocarpa in Obese Rats

Olayinka Anthony Awoyinka^1*, Tolani Rachael², Funmilola Comfort Oladele¹, Margret Olutayo Alese³, Elijah Olalekan Odesanmi⁴, Daisi David Ajayi⁵, Gbenga Sunday Adeleye⁶, Bunmi Comfort Boyede²

¹Department of Medical Biochemistry, College of Medicine, Ekiti State University, Ado Ekiti, Nigeria.
²Department of Microbiology, Ekiti State University, Ado Ekiti, Nigeria.
³Department of Anatomy, College of Medicine, Ekiti State University, Ado Ekiti, Nigeria.
⁴Department of Biochemistry, Ekiti State University, Ado Ekiti, Nigeria.
⁵Department of Chemical Pathology, Ekiti State University Teaching Hospital, Ado Ekiti, Nigeria.
⁶Department of Physiology, Ekiti State University, Ado Ekiti, Nigeria.

DOI: 10.4236/aim.2021.112005                

Abstract

The ubiquitous consumption of junk foods has drastically contributed to the exponential rise in the incidence of obesity. Hence, the present study explores the therapeutic effect of selected indigenous wild bean Sphenostylis stenocarpa (Otili) and condiment fermented Parkia biglobosa (Iru) on obese rats. The rats were fed with a high fat diet for four weeks and the gut microbiota was monitored every other day throughout the period of the experiment. Then, the fecal metabolome was analysed by Gas Chromatography Mass Spectroscopy (GC-MS). Although there was a decrease in the mean weight of rats treated with fermented iru compared with those given Otili, it was not statistically significantly (p ≤ 0.05). The organisms identified from the fecal samples of the fermented Iru groups are Proteus vulgaris, Bacillus cereus and Esherichia coli while those identified from the Otili group include Escherichia coli, and Citrobacter Freundii. However, further study revealed that Otili and Iru had a similar faecal metabolome. Medium chain fatty acids, such as Decanoic acid, Octanoic acid, ethyl tetradecanoate, Hexadecanoic acid, Methyl tetradecanoate, 9-Hexadecanoic acid, Hexadecnoic acid, cis-10-Hepadecanoic acid, are the most common compounds found in this study. This suggests the fact that the associated gut microbiota from breakdown of respective food samples must have actively mediated in their roles of ameliorating the effect of obesity.

Keywords

Microbiota, Metabolomes, MCFA, SCFA, Obese

1. Introduction

Current evidence supports the potential role of the human gut microbiota in obesity. Studies have shown that the bacterial composition of gut microbiota differs between obese and lean individuals; and that a Western-style diet which is high in fat and refined carbohydrates may promote increased intestinal bacteria linked to obesity [1]. This raises the question whether altering the microbiota can modulate the risk of obesity or whether knowledge of an individual’s microbiota can be used to develop personalized diets for obesity prevention [2].

The consumption of beans has received increased attention because of the beneficial physiological effects in the prevention and control of broad range of chronic and degenerative diseases such as obesity [3] [4]. Despite this, there is scarce report on African leguminous plants, such as Parkiabiglobosa (African locust beans) and Sphenostylis stenocarpa (African yam bean); locally known as Iru and Otili in Western Nigeria respectively. The seed of the former is always fermented to produce food condiments for flavouring due to its outstanding protein and amino acid composition [5] [6] [7]. Apart from the nutritional values, fermented African locust bean seeds provide dietary fiber, energy, minerals and vitamins [8] [9]. It also improves sensory properties of foods which include the organoleptic characteristics [10] [11]. However, African yam bean (Sphenostylis stenocarpa) Harms is a seed crop, rich in protein, with the potential to contribute to food security [12]. It can be consumed as dry cooked seed or tube. Seeds are usually added to soups, made into sauces, or milled into flour [13] [14]. This yam bean is a very useful crop because of the ability to thrive under extreme conditions, such as high rainfall, acidity and infertile soil and its resistance to several major crop pests [15].

The bacteria in the human gut not only play an important role in digestion, but research indicates that the microbiome could also play a major role in predisposition to obesity [1]. Against this backdrop, this study was aimed at analysing the gut microbiota and faecal metabolomes in response to high fat diet supplemented with fermentedParkia biglobosa and Sphenostylisstenocarpa in Wistar rats with a view to investigate their anti-obesity potentials.

2. Materials and Methods

2.1. Collection and Preparation of Materials

Dry beans of Sphenostylisstenocarpa (Otili) were sourced from the bush of Ado Ekiti environment, authenticated by the Chief Botanist of the Department of Plant Science, Ekiti State University and deposited in the University Herbarium (Voucher number-BU-1010065). The bean was sorted; sun dried for some hours and later blended into a powder form and stored in a tightly sealed container until use.

Unfermented seeds of Parkiabiglobosa were purchased from a market in Ado-Ekiti, Ekiti State. The seeds were identified and authenticated at the Department of Plant Science, Ekiti State University and deposited in the University Herbarium (Voucher Number UHAE 2020063). The method described by Aderibigbe et al. [16] was adopted for the fermentation process. The dried seeds were hand-picked to remove dirt and boiled under pressure for 3 hours. The cooked seeds were dehulled and washed thoroughly to remove the testa. The cotyledons were boiled again for 1 hour. Three hundred grams (300 g) of the boiled substrate were each weighed separately into twenty sterile baking pans. One millimeter (1 ml) of suitably dialyzed starter cultures was used to inoculate each of the baking pans containing the substrate. The inoculated substrate were mixed using flamed spatula and incubated at 35˚C for 36 hours.

2.2. Experimental Animals

Following institutional ethical approval (ORD/ETHICS/AD/043); 50 albino rats were obtained from the Animal House, College of Medicine, Ekiti State University Ado Ekiti. They were maintained under standard laboratory conditions and fed rat chow with water ad libitum. The rats were grouped into experimental and control groups. Hey were fed for five weeks with a food formation of high fat diet and bean as appropriate while the control group was fed with normal rat chow formulated by BioOrganic Feeds (Table 1). Daily food consumption, body weight, behavioral and physiological changes were observed daily for four weeks as shown in Table 2.

Table 1. Animal diet compositions.

Table 2. Experimental design.

2.3. Microbiota Analysis

The microbiota analysis was investigated using dilution streak plate method as described by Satish [17]; 1 g of the fecal sample from each group was weighed and kept in sterile test tubes. This was followed by the addition of 10 ml of sterile distilled water and the feces allowed to dissolve. Then, 1 ml of the suspension was pipetted into sterile test tubes containing 9 ml of sterile distilled water and shaken. This was repeated until dilution of 10¹ to 10 was obtained. Aseptically, already prepared nutrient agar was poured in duplicates into petri dishes and labeled correctly. A loopful from each of dilutions 10⁻³ was streaked on the already prepared nutrient agar and then incubated at a temperature of 37˚C for 24 hours. The morphological characteristics and numbers of the colonies was observed and then sub-cultured on new plates containing nutrient agar for pure isolation of microorganisms.

2.4. GC-MS Analysis of Microbiota Products

About 3 - 4 mg fecal sample, was exposed to acid methanolysis in 1 M HCl in methanol at 80˚C for 1 hour. The specimen was chromatographically separated on a capillary column with the methylsilicone chemically bonded phase HP-5ms Hewlett-Packard. The comparative concentration of all metabolites was calculated against acetic acid as reference expressing the relative proportion of different metabolites, and the results were expressed in ug/ml [18].

3. Results and Discussion

There has been promising prospects with the manipulation of the gut microbiota to facilitate weight loss or prevent obesity in humans [1]. Possible strategies for obesity prevention and/or treatment include targeting the microbiota, in order to restore or modulate its composition through the consumption of live bacteria (probiotics), nondigestible or limited digestible food constituents such as oligosaccharides (prebiotics), or both (synbiotics), or even fecal transplants [19] [20]. Results from this present study (Figure 1) showed rats fed with otili experienced decrease in weight compared to rats mainly fed with normal chow diet. However, combination of fermented iru+ otili+ high fat diet later caused a decrease in weight perhaps as a result of the otili sample present in the food mixture. This finding corroborates report that mice, with a germ-free gut microbiota, are protected against the obesity that develops after consumption of a Western-style,

Figure 1. Curves showing weight of rats taken every other day.

high fat, sugar-rich diet [21] [22]. It is surmise to say that this present study showed otilito be promising in managing some complications associated with obesity such as bodyweight probably due to its influence on the gut microbiota.

Gut microbiome has been identified in the past decade as an important factor involved in obesity, but the magnitude of its contribution to obesity and its related comorbidities is still uncertain.

Olga et al. [23] submitted that obesity is closely related to the structure of intestinal micro-biota. Higher amount of Bacteroidetes i.e. gram negative bacteria in the gut micro-biota are directly connected with a lean phenotype and with obese individuals who lose weight. Also, reports from both human and animal studies have demonstrated that the relative abundance of Bacteroidetes is reduced by high-fat diet [24]. Although other studies have found changes in gut microbial composition in obese individuals; an increase in the Firmicutes:Bacteroidetes ratio in obesity and an increased abundance of Bacteroidetes during weight loss have not been observed consistently [25] [26]. As shown in Table 3, the micro-biota analysis shows morphological characteristics of microorganisms identified. The distinct organisms identified from the fecal samples of the fermented group^a and group^b are Proteus vulgaris,Bacillus cereus andEsherichia coli and those identified from Otili areEscherichia coli andCitrobacter Freundii. These organisms are gut microbes that inhabit many human body sites mostly residing in the gut. The source of the organism Bacillus spp in the fermented group is probably due to the fact that Bacillus subtilis starter culture was used for the fermentation of the locust bean seeds and hence, the consumption of its microbial cells with the milled seeds by the rats during treatment. Bacillus subtilis play

Table 3. Results of microbiota study.

a significant role in the gut because of their high metabolic activity. Hence, the presence of gram positive Bacillus cereus as a part of the gut microbiome may have contributed to the weight loss of the rats.Interestingly, the Otiligroup had distinctive Escherichia coli and Citrobacter freundii, both gram negative bacteria while the control group fed with normal chow diet had gram positive bacteria- Staphylococcus aureus, and Enterobacter aerogens. These corroborate the earlier submission of Evans et al. [24] that gram negative bacteria in the gut micro-biota are directly connected with obese individuals who lose weight.

Emerging evidence based on numerous animal studies has shown that the gut microbiota and its metabolites, particularly Small chain fatty acids (SCFAs), play an important role in obesity [27] [28] [29]. There have been conflicting results regarding the relationship between SCFAs and obesity. While some studies have reported a positive correlation between fecal SCFA concentrations and obesity [30] [31] [32], others have reported a negative relationship [19] [33]. In this present study, the control group (Figure 2) showed short lipids in form of 9-Hexadecanoic acid with highest concentration of 40.46 ug/ml. This was followed by Hexadecanoic acid (27.51 ug/ml), Decanoic acid 21.46 ug/ml) Octanoic acid (18.65 ug/ml), Dodecanoic acid (17.92 ug/ml), and other unsaturated compounds like Eicosanoic acid as a metabolic product. However, butyrate in form of Butanoic acid, Hexanoic acid and some other compounds were not detected. Otili group (Figure 3) was able to produce almost all the compounds and other unsaturated compounds like Heneicosanoic acid; the highest was 9-Hexadecanoic which have the concentration of 99.13 ug/ml followed by Hexadecanoic acid (28.53 ug/ml) but was unable to produce butanoic acid. For Fermented group^a (Figure 4) short lipids in form of 9-Hexadecanoic acid with concentration of 53.83 ug/ml was also found followed by Hexadecanoic acid (26.83 ug/ml), Decanoic acid (21.44 ug/ml), Octanoic acid (18.25 ug/ml) and other unsaturated compounds like Docosanoic acid as metabolic product; however there was no formation of Butyric acid and some other compounds. For Fermented group^b (Figure 5), short lipids in form of Hexadecanoic acid which have the highest concentration of 27.42 ug/ml was found followed by 9-Hexadecanoic acid with the concentration of 18.52 ug/ml but still was unable to produce Butanoic acid. The absence of Butanoic acid (Butyrate) in this present study portends an answer

Figure 2. Graph showing the metabolic product from control group.

Figure 3. Graph showing the metabolic products from Otiligroup.

Figure 4. Standard graph showing the metabolic product of fermented group^a.

Figure 5. Standard graph showing the metabolic product of fermented group^b.

to the question earlier raised by Lena et al. [34] on the link of butyrate with intestinal microbiota and obesity.

4. Conclusion

This study has revealed that otili(Sphenostylisstenocarpa)and Iru (parkiabiglobosa) has a similar faecal metabolome. Thus, showing Iru and Otili are implicated in lipid metabolism suggesting their anti-obesity potential. This demonstrates the increasing benefits of legumes in the diet and offers practical suggestions to aid health care providers in confidently given informed counsel to obese patients on the consumption of these underutilized, but readily available and affordable species of beans, especially in the midst of limited resources in countries such as Nigeria.

Authors’ Contributions

The corresponding author, AOA designed and led the study and Author BBC analyzed the data. Author OTR, AMO designed the protocol and prepared the first draft of the manuscript. Authors ADD, AGS, OFC managed the analyses of the study. Authors AOA and OTR handled the literature searches. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

References

[1]	Cindy, D. (2016) The Gut Microbiome and Its Role in Obesity. Nutrition Today, 51, 167-174. https://doi.org/10.1097/NT.0000000000000167
[2]	Zeevi, D., Korem, T., Zmora, N., Suez, J., Ali Mahdi, J., Matot, E., et al. (2015) Personalized Nutrition by Prediction of Glycemic Responses. Cell, 163, 1079-1094. https://doi.org/10.1016/j.cell.2015.11.001
[3]	Jenkins, A.L. (2007) The Glycemic Index: Looking Back 25 Years. Cereal foods world, 52, 50-53. https://doi.org/10.1094/CFW-52-1-0050
[4]	Chung, H.J., Liu, Q., Pauls, K.P., Fan, M.Z. and Yada, R. (2008) In-Vitro Starch Digestibility and Some Physicochemical Properties of Starch and Flour from Common Beans. Food Research International, 41, 869-875. https://doi.org/10.1016/j.foodres.2008.03.013
[5]	Cook, J.A., Vanderjagt, D.J., Pastuszyn, A., Mounkaila, G., Glew, R.S., Millson, M. and Glew, R.S. (2000) Nutrients and Chemical Composition of 13 Wild Plant Foods of Niger. Journal of food Composition and Analysis, 13, 83-92. https://doi.org/10.1006/jfca.1999.0843
[6]	Ademola, I.T., Baiyewu, R.A., Adekunle, E.A., Omidiran, M.B. and Adebawo, F.G. (2011) An Assessment into Physical and Proximate Analysis of Processed Locust Bean (Parkia biglobosa) Preserved with Common Salt. Pakistan Journal of Nutrition, 10, 405 408. https://doi.org/10.3923/pjn.2011.405.408
[7]	Uaboi-Egbenni, P.O., Okolie, P.N., Sobande, A.O., Alao, O., Teniola, O. and Bessong, P.O. (2009) Identification of Subdominant Lactic Acid Bacteria in Dawadawa (a Soup Condiment) and Their Evolution during Laboratory-Scale Fermentation of Parkia biglobosa (African Locust Beans). African Journal of Biotechnology, 8, 7241-7248.
[8]	Abdoulaye, O. (2012) Influence of Process Condition on the Digestibility of African Locust Bean (Parkia biglobosa) Starch. American Journal of Food Technology, 7, 552-561. https://doi.org/10.3923/ajft.2012.552.561
[9]	Gbolahan, S. (2013). Biochemical and Chemical Changes during the Fermentation of African Locust Bean Seeds. Chemical Engineering Covenant University, Ota.
[10]	Kolapo, A.L., Popoola, T.O.S., Sanni, M.O. and Afolabi, R.O. (2007) Preservation of Soybean Daddawa Condiment with Dichloromethane Extract of Ginger. Research Journal of Microbiology, 2, 254-259. https://doi.org/10.3923/jm.2007.254.259
[11]	Oladunmoye, M.K. (2007) Effects of Fermentation on Nutrient Enrichment of Locust Beans (Parkia biglobosa, Robert Bam). Research Journal of Microbiology, 2, 185-189.
[12]	Potter, D. and Doyle, J.J. (1992) Origin of African Yam Bean (Sphenostylis stenocarpa, Leguminosae): Evidence from Morphology, Isozymes, Chloroplast DNA and Linguistics. Ecological Botany, 46, 276-292. https://doi.org/10.1007/BF02866626
[13]	Rachie, K.O. and Roberts, L.M. (1974) Grain Legumes of the Low Land Tropics. Advanced Agronomy, 26, 1-132. https://doi.org/10.1016/S0065-2113(08)60869-X
[14]	Nwokolo, E.A. (1987) Nutrient Assessment of African Yam Bean (Sphenostylis stenocarpa) and bambara Groundnut (Voandzea subterranea). Journal of Science Food Agriculture, 41, 123-129. https://doi.org/10.1002/jsfa.2740410205
[15]	Adewale, D. and Dumet, D. (2010) African Yam Bean: A Crop with Food Security Potentials for Africa. Africa Technical Development Forum Journal, 6, 66-67.
[16]	Aderibigbe, E.Y., Visessanguan, W., Somphop, B., Yutthana, K. and Jureeporn, D. (2014) Sourcing Starter Cultures for Parkia biglobosa Fermentation Part II: Potential of Bacillus subtillis Strains. British Microbiology Research Journals, 4, 220-230. https://doi.org/10.9734/BMRJ/2014/6471
[17]	Satish, G. (2004) Short Textbook of Medical Laboratory for Technicians. Jaypee Brothers Medical Publishers Private Limited, New Delhi, 98-99.
[18]	Zamora-Gasga, V.M., Loarca-Piña, G., Vazquez-Landaverde, P.A., Ortiz-Basurto, R.I., Tovar, J. and Sayago-Ayerdi, S.G. (2015) In-Vitro Colonic Fermentation of Food Ingredients Isolated from Agave tequilana Weber var. azul Applied on Granola Bars. Food Science and Technology, 60, 766-772. https://doi.org/10.1016/j.lwt.2014.10.032
[19]	Kim, K.N., Yao, Y. and Ju, S.Y. (2019) Short Chain Fatty Acids and Fecal Microbiota Abundance in Humans with Obesity: A Systematic Review and Meta-Analysis. Nutrients, 11, 2512. https://doi.org/10.3390/nu11102512
[20]	den Besten, G., Lange, K., Havinga, R., van Dijk, T.H., Gerding, A., van Eunen, K., Muller, M., Groen, A.K., Hooiveld, G.J. and Bakker, B.M. (2013) Gut-Derived Short-Chain Fatty Acids Are Vividly Assimilated into Host Carbohydrates and Lipids. American Journal of Physiology-Gastrointestinal and Liver Physiology, 305, G900-G910. https://doi.org/10.1152/ajpgi.00265.2013
[21]	Ley, R.E., Backhed, F., Turnbaugh, P., Lozupone, C.A., Knight, R.D. and Gordon, J.I. (2005) Obesity Alters Gut Microbial Ecology. Proceedings of the National Academy of Sciences of the United States of America, 102, 11070-11075. https://doi.org/10.1073/pnas.0504978102
[22]	Bajzer, M. and Seeley, R.J. (2006) Physiology: Obesity and Gut Flora. Nature, 444, 1009-1010. https://doi.org/10.1038/4441009a
[23]	Olga, C., Albert, G., Park, Y.-M., Seung-Hwan, L., Faidon, M., Sue-Anne, T., Ee, S. and Helmut, S. (2018) The Gut Microbiome Profile in Obesity: A Systematic Review. International Journal of Endocrinology, 2018, Article ID: 6015278. https://doi.org/10.1155/2018/6015278
[24]	Evans, C.C., LePard, K.J., Kwak, J.W., Stancukas, M.C., Laskowski, S., Dougherty, J., Moulton, L., Glawe, A., Wang, Y. and Leone, V. (2014) Exercise Prevents Weight Gain and Alters the Gut Microbiota in a Mouse Model of High Fat Diet-Induced Obesity. PLoS ONE, 9, e92193. https://doi.org/10.1371/journal.pone.0092193
[25]	Zhang, H., DiBaise, J.K., Zuccolo, A., Kudrna, D., Braidotti, M., Yu, Y., et al. (2009) Human Gut Microbiota in Obesity and after Gastric Bypass. Proceedings of the National Academy of Sciences of the United States of America, 106, 2365-2370. https://doi.org/10.1073/pnas.0812600106
[26]	Duncan, S.H., Lobley, G.E., Holtrop, G., Ince, J., Johnstone, A.M., Louis, P., et al. (2008) Human Colonic Microbiota Associated with Diet, Obesity and Weight Loss. International Journal of Obesity, 32, 1720-1724. https://doi.org/10.1038/ijo.2008.155
[27]	Bäckhed, F., Ding, H., Wang, T., Hooper, L.V., Koh, G.Y., Nagy, A., et al. (2004) The Gut Microbiota as an Environmental Factor That Regulates Fat Storage. Proceedings of the National Academy of Sciences of the United States of America, 101, 15718-15723. https://doi.org/10.1073/pnas.0407076101
[28]	Liou, A.P., Paziuk, M., Luevano Jr., J.M., Machineni, S., Turnbaugh, P.J. and Kaplan, L.M. (2013) Conserved Shifts in the Gut Microbiota Due to Gastric Bypass Reduce Host Weight and Adiposity. Science Translational Medicine, 5, 178ra41. https://doi.org/10.1126/scitranslmed.3005687
[29]	Perry, R.J., Peng, L., Barry, N.A., Cline, G.W., Zhang, D., Cardone, R.L., Petersen, K.F., Kibbey, R.G., Goodman, A.L. and Shulman, G.I. (2016) Acetate Mediates a Microbiome-Brain-Beta-Cell Axis to Promote Metabolic Syndrome. Nature, 534, 213-217. https://doi.org/10.1038/nature18309
[30]	Fernandes, J., Su, W., Rahat-Rozenbloom, S., Wolever, T.M. and Comelli, E.M. (2014) Adiposity, Gut Microbiota and Faecal Short Chain Fatty Acids Are Linked in Adult Humans. Nutrition & Diabetes, 4, e121. https://doi.org/10.1038/nutd.2014.23
[31]	Rahat-Rozenbloom, S., Fernandes, J., Gloor, G.B. and Wolever, T.M. (2014) Evidence for Greater Production of Colonic Short-Chain Fatty Acids in Overweight than Lean Humans. International Journal of Obesity, 38, 1525-1531. https://doi.org/10.1038/ijo.2014.46
[32]	Riva, A., Borgo, F., Lassandro, C., Verduci, E., Morace, G., Borghi, E. and Berry D. (2017) Pediatric Obesity Is Associated with an Altered Gut Microbiota and Discordant Shifts in Firmicutes Populations. Environmental Microbiology, 19, 95-105. https://doi.org/10.1111/1462-2920.13463
[33]	Muhammad, J.K., Konstantinos, G., Christine, A.E. and Guftar, M.S. (2016) Role of Gut Microbiota in the Aetiology of Obesity. Journal of Obesity, 2016, Article ID: 7353642.
[34]	Lena, K.B., Arne, A. and Lesli, L. (2013) Is Butyrate the Link between Diet, Intestinal Microbiota and Obesity-Related Metabolic Diseases? Obesity Reviews, 14, 950-959. https://doi.org/10.1111/obr.12068
[35]	Schwiertz, A., Taras, D., Schafer, K., Beijer, S., Bos, N.A., Donus, C. and Hardt, P.D. (2010) Microbiota and SCFA in Lean and Overweight Healthy Subjects. Obesity, 18, 190-195. https://doi.org/10.1038/oby.2009.167
[36]	Barczynska, R., Litwin, M., Slizewska, K., Szalecki, M., Berdowska, A., Bandurska, K., Libudzisz, Z. and Kapusniak, J. (2018) Bacterial Microbiota and Fatty Acids in the Faeces of Overweight and Obese Children. Polish Journal of Microbiology, 67, 339-345. https://doi.org/10.21307/pjm-2018-041