Effect of Spices on the Microbial Diversity, Physicochemical Properties and Nutritional Properties of Fermented Millet (Pennisetum glaucum) Slurry

https://doi.org/10.51867/ajernet.6.2.2

Authors

Keywords:

Bacteria Diversity, Fermentation, Millet, Lactic Acid, Nutrition, Spices

Abstract

Fermentation improves taste, adds flavour and makes nutrients easily digestible. Millet porridge is produced from spontaneous fermentation of pearl millet grain with or without spices. A study was carried out to evaluate the proximate, nutritional, and bacterial diversity composition during fermentation of pearl millet slurry with and without spices. In this experimental study, some millet grains were fermented with selected spices, while others were fermented without spices. The pH, proximate and mineral analysis, and lactic acid production were determined in the fermented products. The 16S ribosomal RNA (16S rRNA) metagenomic method was used to identify the microbial diversity and abundance in the fermented millet slurry with and without spices. The slurry fermented with spices recorded 22.50 (mg/g) iron at 24 hours whilst without spices recorded a 10.10 (mg/g) iron content. Furthermore, zinc content at 24 hours for slurry with spices was 175.50 (µg/g) whilst without spices was 60.10 (µg/g). The lactic acid content for millet slurry without spices was between 7.16µg/mL and 9.22µg/mL whilst the lactic acid content for millet slurry with spices was between 6.55µg/mL and 9.88µg/mL produced after a 72-h fermentation period. Acetobacter was the most dominant genera in the fermented slurry (54.23%). The relative abundance of the genus Lacticaseibacillus (19.71% to 1.69%), Lactobacillus (0.49% to 5.25%), Limosilactobacillus (2.43% to 40.96%), Acetobacter (54.23% to 34.93%), Schleiferilactobacillus (16.29% to 0.84%) were present in the fermented slurry. The fermentation of pearl millet grains with spices improves the nutritional composition of pearl millet and provides a diversified fermenting bacteria community. The most dominant species in the slurry fermentation can be formulated into starter cultures to be used in controlled fermentation.

Dimensions

Abedi, E., & Hashemi, S. M. B. (2020). Lactic acid production-producing microorganisms and substrates sources-state of art. Heliyon, 6(10), e04974. https://doi.org/10.1016/j.heliyon.2020.e04974 DOI: https://doi.org/10.1016/j.heliyon.2020.e04974

Adebiyi, J., Obadina, A., Adebo, O., & Kayitesi, E. (2018). Fermented and malted millet products in Africa: Expedition from traditional/ethnic foods to industrial value-added products. Critical Reviews in Food Science and Nutrition, 58(3), 463-474. https://doi.org/10.1080/10408398.2016.1186123

Adebo, J. A., Njobeh, P. B., Gbashi, S., Oyedeji, A. B., Ogundele, O. M., Oyeyinka, S. A., & Adebo, O. A. (2022). Fermentation of cereals and legumes: Impact on nutritional constituents and nutrient bioavailability. Fermentation, 8(2), 63.

https://doi.org/10.3390/fermentation8020063 DOI: https://doi.org/10.3390/fermentation8020063

Adebo, O. A., & Medina-Meza, I. G. (2020). Impact of fermentation on the phenolic compounds and antioxidant activity of whole cereal grains: A mini review. Molecules, 25(4), 927. https://doi.org/10.3390/molecules25040927 DOI: https://doi.org/10.3390/molecules25040927

Adejobi, T., Johnson, O., Omolara, A., Oguntoyinbo, O., & Enujiugha, V. (2024). Effect of ginger and garlic inclusion on the performance of Lactobacillus plantarum in maize (Zea mays L.) fermentation into Ogi. IPS Journal of Applied Microbiology and Biotechnology, 3(1), 46-56.

https://doi.org/10.54117/ijamb.v3i1.18 DOI: https://doi.org/10.54117/ijamb.v3i1.18

Adelekan, A., & Alamu, A. (2021). Effect of enrichment with turmeric and ginger on some quality characteristics of fermented maize Ogi. Croatian Journal of Food Science and Technology, 13(2), 1-11. https://doi.org/10.17508/CJFST.2021.13.2.11 DOI: https://doi.org/10.17508/CJFST.2021.13.2.11

Adisa, A., Bola Adepeju, A., Adisa, A. M., Adepeju, A. B., & Yusuf, A. K. (2019). Influence of pH and acidity on the fermentation of finger millet spiced Ogi. Journal of Faculty of Food Engineering, 18(3), 214-222.

AOAC. (2002). Official method of analysis (17th ed.). Maryland, USA.

AOAC. (2005). Official method of analysis (18th ed.). Maryland, USA.

Assohoun, M. C., Djeni, T. N., Koussémon-Camara, M., & Brou, K. (2013). Effect of fermentation process on nutritional composition and aflatoxins concentration of Doklu, a fermented maize-based food. Food and Nutrition Sciences, 4(11), 1120. DOI: https://doi.org/10.4236/fns.2013.411146

https://doi.org/10.4236/fns.2013.411144 DOI: https://doi.org/10.4236/fns.2013.411144

Atter, A., Diaz, M., Tano-Debrah, K., Kunadu, A. P.-H., Mayer, M. J., Colquhoun, I. J., Nielsen, D. S., Baker, D., Narbad, A., & Amoa-Awua, W. (2021). Microbial diversity and metabolite profile of fermenting millet in the production of Hausa Koko, a Ghanaian fermented cereal porridge. Frontiers in Microbiology, 12, 681983.

https://doi.org/10.3389/fmicb.2021.681983 DOI: https://doi.org/10.3389/fmicb.2021.681983

Baev, V., Apostolova, E., Gotcheva, V., Koprinarova, M., Papageorgiou, M., Rocha, J. M., Yahubyan, G., & Angelov, A. (2023). 16S-rRNA-based metagenomic profiling of the bacterial communities in traditional Bulgarian sourdoughs. Microorganisms, 11(3), 803. https://doi.org/10.3390/microorganisms11030803 DOI: https://doi.org/10.3390/microorganisms11030803

Banwo, K., Asogwa, F. C., Ogunremi, O. R., Adesulu-Dahunsi, A., & Sanni, A. (2021). Nutritional profile and antioxidant capacities of fermented millet and sorghum gruels using lactic acid bacteria and yeasts. Food Biotechnology, 35(3), 199-220.

https://doi.org/10.1080/08905436.2021.1940197 DOI: https://doi.org/10.1080/08905436.2021.1940197

Borquaye, L. S., Darko, G., Laryea, M. K., Gasu, E. N., Amponsah, N. A. A., & Appiah, E. N. (2017). Nutritional and anti-nutrient profiles of some Ghanaian spices. Cogent Food & Agriculture, 3(1), 1348185. https://doi.org/10.1080/23311932.2017.1348185 DOI: https://doi.org/10.1080/23311932.2017.1348185

Borshchevskaya, L. N., Gordeeva, T. L., Kalinina, A. N., & Sineokii, S. P. (2016). Spectrophotometric determination of lactic acid. Journal of Analytical Chemistry, 71(8), 755-758. https://doi.org/10.1134/S1061934816080037 DOI: https://doi.org/10.1134/S1061934816080037

Budhwar, S., Sethi, K., & Chakraborty, M. (2020). Efficacy of germination and probiotic fermentation on underutilized cereal and millet grains. Food Production, Processing and Nutrition, 2(12), 1-17. https://doi.org/10.1186/s43014-020-00026-w DOI: https://doi.org/10.1186/s43014-020-00026-w

Church, D. L., Cerutti, L., Gürtler, A., Griener, T., Zelazny, A., & Emler, S. (2020). Performance and application of 16S rRNA gene cycle sequencing for routine identification of bacteria in the clinical microbiology laboratory. Clinical Microbiology Reviews, 33(4), e00053-19. https://doi.org/10.1128/CMR.00053-19 DOI: https://doi.org/10.1128/CMR.00053-19

Coelho, L., De Lima, C., Rodovalho, C., Bernardo, M., & Contiero, J. (2011). Lactic acid production by new Lactobacillus plantarum LMISM6 grown in molasses: Optimization of medium composition. Brazilian Journal of Chemical Engineering, 28(8), 27-36. https://doi.org/10.1590/S0104-66322011000100004 DOI: https://doi.org/10.1590/S0104-66322011000100004

Desta, D. T., Kelikay, G. N., Zekwos, M., Eshete, M., Reda, H. H., Alemayehu, F. R., & Zula, A. T. (2021). Influence of fermentation time on proximate composition and microbial loads of Enset (Ensete ventricosum), sampled from two different agroecological districts. Food Science & Nutrition, 9(10), 5641-5647. https://doi.org/10.1002/fsn3.2527 DOI: https://doi.org/10.1002/fsn3.2527

Diaz, M., Kellingray, L., Akinyemi, N., Adefiranye, O. O., Olaonipekun, A. B., Bayili, G. R., Ibezim, J., Du Plessis, A. S., Houngbédji, M., & Kamya, D. (2019). Comparison of the microbial composition of African fermented foods using amplicon sequencing. Scientific Reports, 9(1), 13863. https://doi.org/10.1038/s41598-019-50190-4 DOI: https://doi.org/10.1038/s41598-019-50190-4

El-Askri, T., Yatim, M., Sehli, Y., Rahou, A., Belhaj, A., Castro, R., Durán-Guerrero, E., Hafidi, M., & Zouhair, R. (2022). Screening and characterization of new Acetobacter fabarum and Acetobacter pasteurianus strains with high ethanol-thermo tolerance and the optimization of acetic acid production. Microorganisms, 10(9), 1741. https://doi.org/10.3390/microorganisms10091741 DOI: https://doi.org/10.3390/microorganisms10091741

Feiner, G. (2006). The microbiology of specific bacteria. In G. Feiner (Ed.), Meat Products Handbook (pp. 595-615). Woodhead Publishing. https://doi.org/10.1533/9781845691721.3.595 DOI: https://doi.org/10.1533/9781845691721.3.595

Gabaza, M., Muchuweti, M., Vandamme, P., & Raes, K. (2017). Can fermentation be used as a sustainable strategy to reduce iron and zinc binders in traditional African fermented cereal porridges or gruels? Food Reviews International, 33(6), 561-586. https://doi.org/10.1080/87559129.2016.1196491 DOI: https://doi.org/10.1080/87559129.2016.1196491

Gupta, R., & Meghwal, M. (2021). Fermented food based on cereal and pulses. In Advances in Cereals Processing Technologies (pp. 153-217). CRC Press. https://doi.org/10.1201/9781003261124-8 DOI: https://doi.org/10.1201/9781003261124-8

Hassan, Z. M., Sebola, N. A., & Mabelebele, M. (2021). The nutritional use of millet grain for food and feed: A review. Agriculture and Food Security, 10(1), 1-14.

https://doi.org/10.1186/s40066-020-00282-6 DOI: https://doi.org/10.1186/s40066-020-00282-6

Ibrahim, R., Shukri, N. S. M., & Mazidi, M. N. I. H. (2020). Effect of fresh spices on the fermentation, colonization of lactic acid bacteria and sensory acceptability of fermented clams (Mercenaria mercenaria). IOP Conference Series: Materials Science and Engineering. https://doi.org/10.1088/1757-899X/932/1/012004 DOI: https://doi.org/10.1088/1757-899X/932/1/012004

Inglis, L. K., & Edwards, R. A. (2022). How metagenomics has transformed our understanding of bacteriophages in microbiome research. Microorganisms, 10(8).

https://doi.org/10.3390/microorganisms10081671 DOI: https://doi.org/10.3390/microorganisms10081671

Inyang, C., & Zakari, U. (2008). Effect of germination and fermentation of pearl millet on proximate, chemical and sensory properties of instant "Fura"-a Nigerian cereal food. Pakistan Journal of Nutrition, 7(1), 9-12. https://doi.org/10.3923/pjn.2008.9.12 DOI: https://doi.org/10.3923/pjn.2008.9.12

Kambabazi, M. R., Hitayezu, E., & Mukandahiro, Y. (2019). Assessment of microbiological changes during production of malted and fermented finger millet flour. Rwanda Journal of Agricultural Sciences, 1(1), 31-35.

Khan, U. S. (2014). Probiotics in dairy foods: A review. Nutrition & Food Science, 44(1), 71-88. https://doi.org/10.1108/NFS-04-2013-0051 DOI: https://doi.org/10.1108/NFS-04-2013-0051

Kharat, S., Medina-Meza, I. G., Kowalski, R. J., Hosamani, A., Ramachandra, C., Hiregoudar, S., & Ganjyal, G. M. (2019). Extrusion processing characteristics of whole grain flours of select major millets (foxtail, finger, and pearl). Food and Bioproducts Processing, 114, 60-71. https://doi.org/10.1016/j.fbp.2018.07.002 DOI: https://doi.org/10.1016/j.fbp.2018.07.002

Klindworth, A., Pruesse, E., Schweer, T., Peplies, J., Quast, C., Horn, M., & Glöckner, F. O. (2012). Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Research, 41(1), e1-e1. https://doi.org/10.1093/nar/gks808 DOI: https://doi.org/10.1093/nar/gks808

Krishnan, R., & Meera, M. (2018). Pearl millet minerals: Effect of processing on bioaccessibility. Journal of Food Science and Technology, 55(7), 3362-3372.

https://doi.org/10.1007/s13197-018-3305-9 DOI: https://doi.org/10.1007/s13197-018-3305-9

Kumar, A., Rani, M., Mani, S., Shah, P., Singh, D. B., Kudapa, H., & Varshney, R. K. (2021). Nutritional significance and antioxidant-mediated antiaging effects of finger millet: Molecular insights and prospects. Frontiers in Sustainable Food Systems, 5, 1-17. https://doi.org/10.3389/fsufs.2021.684318 DOI: https://doi.org/10.3389/fsufs.2021.684318

Li, S., Dong, X., Fan, G., Yang, Q., Shi, J., Wei, W., Zhao, F., Li, N., Wang, X., & Wang, F. (2018). Comprehensive profiling and inheritance patterns of metabolites in foxtail millet. Frontiers in Plant Science, 9, 1716. https://doi.org/10.3389/fpls.2018.01716 DOI: https://doi.org/10.3389/fpls.2018.01716

Lu, Q. Y., Rasmussen, A. M., Yang, J., Lee, R. P., Huang, J., Shao, P., & Li, Z. (2019). Mixed spices at culinary doses have prebiotic effects in healthy adults: A pilot study. Nutrients, 11(6), 1-14. https://doi.org/10.3390/nu11061425

https://doi.org/10.3390/nu11061425 DOI: https://doi.org/10.3390/nu11061425

Lund, P. A., De Biase, D., Liran, O., Scheler, O., Mira, N. P., Cetecioglu, Z., Fernández, E. N., Bover-Cid, S., Hall, R., & Sauer, M. (2020). Understanding how microorganisms respond to acid pH is central to their control and successful exploitation. Frontiers in Microbiology, 11, 556140. https://doi.org/10.3389/fmicb.2020.556140 DOI: https://doi.org/10.3389/fmicb.2020.556140

Manjula, K., Bhagath, Y. B., & Nagalakshmi, K. (2015). Effect of radiation processing on bioactive components of finger millet flour (Eleusine coracana L.). International Food Research Journal, 22(2), 556-560.

Mishra, P., & Sabikhi, L. (2020). Fermented pearl millet weaning food: An innovation of food technology and application in food processing and management. In P. Mishra, R. R. Mishra, & C. O. Adetunji (Eds.), Innovations in Food Technology (pp. 17-27). Springer, Singapore. https://doi.org/10.1007/978-981-15-6121-4_2 DOI: https://doi.org/10.1007/978-981-15-6121-4_2

Mutshinyani, M., Mashau, M. E., & Jideani, A. I. O. (2020). Bioactive compounds, antioxidant activity and consumer acceptability of porridges of finger millet (Eleusine coracana) flours: Effects of spontaneous fermentation. International Journal of Food Properties, 23(1), 1692-1710. https://doi.org/10.1080/10942912.2020.1825485 DOI: https://doi.org/10.1080/10942912.2020.1825485

Nkhata, S. G., Ayua, E., Kamau, E. H., & Shingiro, J. B. (2018). Fermentation and germination improve nutritional value of cereals and legumes through activation of endogenous enzymes. Food Science & Nutrition, 6(8), 2446-2458.

https://doi.org/10.1002/fsn3.846 DOI: https://doi.org/10.1002/fsn3.846

Ocheme, O., & Chinma, C. (2008). Effects of soaking and germination on some physicochemical properties of millet flour for porridge production. Journal of Food Technology, 6(5), 185-188.

Olaniran, A., Abiose, S., Adeniran, H., Gbadamosi, S., & Iranloye, Y. (2020). Production of a cereal-based product (Ogi): Influence of co-fermentation with powdered garlic and ginger on the microbiome. Agrosearch, 20(1), 81-93.

https://doi.org/10.4314/agrosh.v20i1.8S DOI: https://doi.org/10.4314/agrosh.v20i1.8S

Onweluzo, J., & Nwabugwu, C. (2009). Fermentation of millet (Pennisetum americanum) and pigeon pea (Cajanus cajan) seeds for flour production: Effects on composition and selected functional properties. Pakistan Journal of Nutrition, 8(6), 737-744. https://doi.org/10.3923/pjn.2009.737.744 DOI: https://doi.org/10.3923/pjn.2009.737.744

Osman, M. A. (2011). Effect of traditional fermentation process on the nutrient and antinutrient contents of pearl millet during preparation of Lohoh. Journal of the Saudi Society of Agricultural Sciences, 10(1), 1-6.

https://doi.org/10.1016/j.jssas.2010.06.001 DOI: https://doi.org/10.1016/j.jssas.2010.06.001

Ramashia, S. E., Anyasi, T. A., Gwata, E. T., Meddows-Taylor, S., & Jideani, A. I. O. (2019). Processing, nutritional composition and health benefits of finger millet in Sub-Saharan Africa. Food Science and Technology, 39(2), 253-266. https://doi.org/10.1590/fst.25017 DOI: https://doi.org/10.1590/fst.25017

Sahlin, P., & Nair, B. M. (2012). Production of organic acids, titratable acidity and pH-development during fermentation of cereal flours. International Journal of Fermented Foods, 1(1), 15-32.

Saini, S., Saxena, S., Samtiya, M., & Puniya, M. (2021). Potential of underutilized millets as nutri-cereal: An overview. Journal of Food Science and Technology. https://doi.org/10.1007/s13197-021-04985-x DOI: https://doi.org/10.1007/s13197-021-04985-x

Saleh, A. S. M., Zhang, Q., Chen, J., & Shen, Q. (2013). Millet grains: Nutritional quality, processing, and potential health benefits. Comprehensive Reviews in Food Science and Food Safety, 12(3), 281-295. https://doi.org/10.1111/1541-4337.12012 DOI: https://doi.org/10.1111/1541-4337.12012

Samtiya, M., Aluko, R. E., Puniya, A. K., & Dhewa, T. (2021). Enhancing micronutrients bioavailability through fermentation of plant-based foods: A concise review. Fermentation, 7(2), 63. https://doi.org/10.3390/fermentation7020063 DOI: https://doi.org/10.3390/fermentation7020063

Sarita, & Singh, E. (2016). Millet's anti-nutrients and their therapeutic effects. The Pharma Journal, 5(8), 42-46.

Sarita, E. S. (2016). Potential of millets: Nutrients composition and health benefits. Journal of Scientific & Innovative Research, 5(2), 46-50.

https://doi.org/10.31254/jsir.2016.5204 DOI: https://doi.org/10.31254/jsir.2016.5204

Sauer, S., Dlugosch, L., Milke, F., Brinkhoff, T., Kammerer, D. R., Stintzing, F. C., & Simon, M. (2022). Succession of bacterial and fungal communities during fermentation of medicinal plants. Fermentation, 8(8), 383.

https://doi.org/10.3390/fermentation8080383 DOI: https://doi.org/10.3390/fermentation8080383

Serba, D. D., Yadav, R. S., Varshney, R. K., Gupta, S., Mahalingam, G., Srivastava, R. K., Gupta, R., Perumal, R., & Tesso, T. T. (2020). Genomic designing of pearl millet: A resilient crop for arid and semi-arid environments. In C. Kole (Ed.), Genomic designing of climate-smart cereal crops (pp. 221-286). Springer.

https://doi.org/10.1007/978-3-319-93381-8_6 DOI: https://doi.org/10.1007/978-3-319-93381-8_6

Sharma, R., Garg, P., Kumar, P., Bhatia, S. K., & Kulshrestha, S. (2020). Microbial fermentation and its role in quality improvement of fermented foods. Fermentation, 6(4), 106. https://doi.org/10.3390/fermentation6040106 DOI: https://doi.org/10.3390/fermentation6040106

Sharma, S., Jain, S., Nair, G. N., & Srinivasan, R. (2013). Capsicum annuum enhances L-lactate production by Lactobacillus acidophilus: Implication in curd formation. Journal of Dairy Science, 96(7), 4142-4148. https://doi.org/10.3168/jds.2012-6243 DOI: https://doi.org/10.3168/jds.2012-6243

Sinclair, L., Osman, O. A., Bertilsson, S., & Eiler, A. (2015). Microbial community composition and diversity via 16S rRNA gene amplicons: Evaluating the Illumina platform. PLoS ONE, 10(2), e0116955. https://doi.org/10.1371/journal.pone.0116955 DOI: https://doi.org/10.1371/journal.pone.0116955

Singh, J., Behal, A., Singla, N., Joshi, A., Birbian, N., Singh, S., Bali, V., & Batra, N. (2009). Metagenomics: Concept, methodology, ecological inference and recent advances. Biotechnology journal, 4(4), 480-494. https://doi.org/10.1002/biot.200800201 DOI: https://doi.org/10.1002/biot.200800201

Tang, J., Wang, X. C., Hu, Y., Zhang, Y., & Li, Y. (2017). Effect of pH on lactic acid production from acidogenic fermentation of food waste with different types of inocula. Bioresource Technology, 224, 544-552. https://doi.org/10.1016/j.biortech.2016.11.111 DOI: https://doi.org/10.1016/j.biortech.2016.11.111

Taylor, J. R. N., & Duodu, K. G. (2014). Effects of processing sorghum and millets on their phenolic phytochemicals and the implications of this to the health-enhancing properties of sorghum and millet food and beverage products. J Sci Food Agric, 95(5), 225-237. https://doi.org/10.1002/jsfa.6713 DOI: https://doi.org/10.1002/jsfa.6713

Thomas, T., Gilbert, J., & Meyer, F. (2012). Metagenomics - A guide from sampling to data analysis. Microbial Informatics and Experimentation, 2(3), 1-13. https://doi.org/10.1186/2042-5783-2-3 DOI: https://doi.org/10.1186/2042-5783-2-3

Tonapi, V. A., Thirunavukkarasu, N., Gupta, S., Gangashetty, P. I., & Yadav, O. (2024). Pearl millet in the 21st century. Springer. https://doi.org/10.1007/978-981-99-5890-0 DOI: https://doi.org/10.1007/978-981-99-5890-0

Wang, X., Zhang, P., & Zhang, X. (2021). Probiotics regulate gut microbiota: An effective method to improve immunity. Molecules, 26(19), 6076. https://doi.org/10.3390/molecules26196076 DOI: https://doi.org/10.3390/molecules26196076

Youseif, S. H., Abd El-Megeed, F. H., Humm, E. A., Maymon, M., Mohamed, A. H., Saleh, S. A., & Hirsch, A. M. (2021). Comparative analysis of the cultured and total bacterial community in the wheat rhizosphere microbiome using culture-dependent and culture-independent approaches. Microbiology Spectrum, 9(2), e00678-00621. https://doi.org/10.1128/Spectrum.00678-21 DOI: https://doi.org/10.1128/Spectrum.00678-21

Zhang, Z., Ping, Q., Gao, D., Vanrolleghem, P. A., & Li, Y. (2021). Effects of ferric-phosphate forms on phosphorus release and the performance of anaerobic fermentation of waste activated sludge. Bioresource Technology, 323, 124622.

https://doi.org/10.1016/j.biortech.2020.124622 DOI: https://doi.org/10.1016/j.biortech.2020.124622

Zhao, L., Mo, X., Zhang, C., Yang, L., & Wang, X. (2021). Community diversity and succession in fermented grains during the stacking fermentation of Chinese moutai-flavored liquor making. Food Science and Technology, 42, e61521. https://doi.org/10.1590/fst.61521 DOI: https://doi.org/10.1590/fst.61521

Published

2025-04-03

How to Cite

Osei-Bimpong, B., Agyirifo, D. S., Mensah, T. A., Tepson, J. A., & Adotey, J. P. (2025). Effect of Spices on the Microbial Diversity, Physicochemical Properties and Nutritional Properties of Fermented Millet (Pennisetum glaucum) Slurry. African Journal of Empirical Research, 6(2), 11–25. https://doi.org/10.51867/ajernet.6.2.2