Fermentation of WildHydrolytic Bacteria Isolated from Opuntia ficus-indica, and Its Effecton Flow Behavior of Cladodes Flour Culture Media

Abstract

The aim of this studywas to evaluate the fermentation dynamics of two wild hydrolytic microorganismsand their effect on the flow behavior of a culture medium formulated with Opuntiaficus-indica cladode flour. Identified Acinetobacter pitti and Bacillus subtilis presented maximumvalues of specific hydrolytic activity (SHA) at 24 h of growth (0.21 ± 0.05 and 0.22 ± 0.01 IU,respectively). The apparent viscosity of cladode flour medium (CFM) measured byapplying shear rates (66.7 s^-1-0.003 s^-1) in suspensions (20%) showed asignificant decrease (60%) as afunction of bacterial growth progressed. After fermentation, the CFM exhibitedpseudoplastic (shear-thinning) behavior, which was linked to the enzymaticdegradation of polysaccharides. The use of crude extracellular enzyme extractsfrom these wild bacteria effectively reduced medium viscosity by breaking downthe plant matrix. These findings highlightthe hydrolytic potential of native strains in modifying the rheologicalproperties of cactus-based culture media, offering a low-cost alternative forbiomass pretreatment and valorization in future biotechnological applications.

Keywords

Opuntia ficus-indica / Acinetobacterpittii / Kluyveromyces marxianus / Cladodes / Flow behavior

Author Contributions

Conceptualization, M.O.R.-S., I.M.R.-B. and C.M.L.-D.; Methodology, I.M.R.-B. and C.M.L.-D.; Software, C.M.L.-D. and K.A.A.-B.; Validation, M.O.R.-S., I.M.R.-B. and C.M.L.-D.; Formal Analysis, M.O.R.-S. and I.M.R.-B; Investigation, I.M.R.-B. and K.A.A.-B.; Resources, M.O.R.-S.; Data Curation, C.M.L.-D.; Writing—Original Draft Preparation, M.O.R.-S. and C.M.L.-D.; Writing—Review & Editing, C.M.L.-D., K.A.A.-B. and I.M.R.-B.; Visualization, M.O.R.-S.; Supervision, M.O.R.-S., I.M.R.-B.; Project Administration, M.O.R.-S., I.M.R.-B.; Funding Acquisition, I.M.R.-B.

Ethics Statement

Not applicable.

Data Availability Statement

The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Funding

This work was supported by the National Council of Science and Technology of Mexico (CONACYT) and Secretariat of Agriculture, Livestock, Rural Development, Fisheries and Food (SAGARPA) with project No. 195157. The author López-Domínguez thanks to National Council of Science and Technology of Mexico (CONACYT) for scholarship No. 265844.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

[1] Prisa D. Opuntia plants nutritive and medicinal value: A review. J. Curr. Sci. Technol. 2023, 13, 486-499. doi: 10.59796/jcst.V13N2.2023.1762.

[2] SIAP . Servicio de Información Agroalimentaria y Pesquera. Available online: http://www.gob.mx/siap/ (accessed on 10 May 2024).

[3] Bensadón S, Hervert-Hernández D, Sáyago-Ayerdi SG, Goñi I. By-products of Opuntia ficus-indica as a source of antioxidant dietary fiber. Plant Foods Hum. Nutr. 2010, 65, 210-216. doi: 10.1007/s11130-010-0176-2.

[4] SAGARPA . Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación. Available online: https://www.gob.mx/sagarpa (accessed on 13 March 2016).

[5] Bermejo LA, Sánchez-Zapata E, Pérez-Álvarez JA, Viuda-Martos M. Effect of enzymatic treatment on dietary fiber structure and functional properties in cactus pear (Opuntia ficus-indica) peel. Food Hydrocoll. 2022, 129, 107662. doi: 10.1016/j.foodhyd.2022.107662.

[6] Garfias Silva V, Cordova Aguilar MS, Ascanio G, Aguayo JP, Pérez-Salas KY, Susunaga Notario AC. Acid hydrolysis of pectin and mucilage from cactus (Opuntia ficus) for identification and quantification of monosaccharides. Molecules 2022, 27, 5830. doi: 10.3390/molecules27185830.

[7] Rodríguez-González F, Pérez-González J, Muñoz-López CN, Vargas-Solano SV, Marín-Santibáñez BM. Influence of age on molecular characteristics and rheological behavior of nopal mucilage. Food Sci. Nutr. 2021, 9, 6776-6785. doi: 10.1002/fsn3.2629.

[8] Luna-Zapién EA, Zegbe JA, Meza-Velázquez JA, Contreras-Esquivel JC, Morales-Martínez TK. Mucilage yield, composition, and physicochemical properties of cultivated cactus pear varieties as influenced by irrigation. Agronomy 2023, 13, 419. doi: 10.3390/agronomy13020419.

[9] Martins M, Ribeiro MH, Almeida CMM. Physicochemical, nutritional, and medicinal properties of Opuntia ficus-indica (L.) Mill. and its main agro-industrial use: A review. Plants 2023, 12, 1512. doi: 10.3390/plants12071512.

[10] Quintero-García M, Gutiérrez-Cortez E, Bah M, Rojas-Molina A, Cornejo-Villegas MA, Real AD, Rojas-Molina I. Comparative analysis of the chemical composition and physicochemical properties of the mucilage extracted from fresh and dehydrated Opuntia ficus-indica cladodes. Foods 2021, 10, 2137. doi: 10.3390/foods10092137.

[11] León-Martínez FM, Méndez-Lagunas LL, Rodríguez-Ramírez J. Spray drying of nopal mucilage (Opuntia ficus-indica): Effects on powder properties and characterization. Carbohydr. Polym. 2010, 81, 864-870. doi: 10.1016/j.carbpol.2010.03.061.

[12] Sáenz C, Sepúlveda E, Pak N, Lecaros M. Chemical and physical characterization of cactus cladodes (Opuntia ficus-indica) powder. Ital. J. Food Sci. 2010, 22, 416-423.

[13] Medina-Torres L, Brito-De La Fuente E, Torrestiana-Sanchez B, Alonso S. Mechanical properties of gels formed by mixtures of mucilage gum (Opuntia ficus-indica) and carrageenans. Carbohydr. Polym. 2003, 52, 143-150. doi: 10.1016/S0144-8617(02)00269-2.

[14] Rooyen B, de Wit M, Osthoff G, van Niekerk J. Cactus pear mucilage (Opuntia spp.) as a novel functional biopolymer: Mucilage extraction, rheology and biofilm development. Polymers 2024, 16, 1993. doi: 10.3390/polym16141993.

[15] Cai W, Gu X, Tang J. Extraction, purification, and characterization of the polysaccharides from Opuntia milpa alta. Carbohydr. Polym. 2008, 71, 403-410. doi: 10.1016/j.carbpol.2007.06.008.

[16] Fernández-Martínez MC, Jiménez-Martínez C, Jaime-Fonseca MR, Alamilla-Beltrán L. Extraction of purple prickly pear (Opuntia ficus-indica) mucilage by microfiltration, composition, and physicochemical characteristics. Polymers 2024, 16, 3383. doi: 10.3390/polym16233383.

[17] Feugang JM, Konarski P, Zou D. Nutritional and medicinal use of cactus pear (Opuntia spp.) cladodes and fruits. Front Biosci. 2006, 11, 2574-2589.

[18] Gurrieri S, Miceli L, Lanza CM, Tomaselli F, Bonomo RP, Rizzarelli E. Chemical characterization of Sicilian prickly pear (Opuntia ficus-indica) and perspectives for the storage of its juice. J. Agric. Food Chem. 2000, 48, 5424-5431. doi: 10.1021/jf9907844.

[19] Atlas R. Handbook of Microbiological Media, 3rd ed.; CRC Press: Boca Raton, FL, USA, 2004.

[20] AOAC . Official Methods of Analysis, 14th ed.; AOAC: Washington, DC, USA, 1984.

[21] AOAC . Official Methods of Analysis, 18th ed.; AOAC: Gaithersburg, MD, USA, 2005.

[22] Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956, 28, 350-356.

[23] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 1959, 31, 426-428. doi: 10.1021/ac60147a030.

[24] United States Environmental Protection Agency (EPA). Method 200.7: Determination of Metals and Trace Elements in Water and Wastes by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES)(Revision 4.4); U.S. Environmental Protection Agency, Office of Water: Washington, DC, USA, 1994.

[25] Ayadi MA, Abdelmaksoud W, Ennouri M, Attia H. Cladodes from Opuntia ficus-indica as a source of dietary fiber: Effect on dough characteristics and cake making. Ind. Crops Prod. 2009, 30, 40-47. doi: 10.1016/j.indcrop.2009.01.003.

[26] Teather RM, Wood PJ. Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 1982, 43, 777-780. doi: 10.1128/aem.43.4.777-780.1982.

[27] Bartholomew JW, Finkelstein H. Relationship of cell wall staining to gram differentiation. J. Bacteriol. 1958, 75, 77-84.

[28] Hills PN, van Staden J. An improved DNA extraction procedure for plant tissues with a high phenolic content. S. Afr. J. Bot. 2002, 68, 549-550. doi: 10.1016/S0254-6299(15)30384-7.

[29] Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 2012, 28, 1647-1649. doi: 10.1093/bioinformatics/bts199.

[30] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403-410. doi: 10.1016/S0022-2836(05)80360-2.

[31] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254. doi: 10.1016/0003-2697(76)90527-3.

[32] Ramírez F, Demirer G, Gallegos C, Hernández G, Santoyo V, Espinosa T. Life cycle assessment of biogas production through anaerobic co-digestion of nopal cladodes and dairy cow manure. J. Clean Prod. 2018, 172, 2313-2322.

[33] Astello-García MG, Cervantes I, Nair V, Santos-Díaz MS, Reyes-Agüero A, Guéraud F, et al. Chemical composition and phenolic compounds profile of cladodes from Opuntia spp. cultivars with different domestication gradient. J. Food Compos. Anal. 2015, 43, 119-130. doi: 10.1016/j.jfca.2015.04.016.

[34] Blanco-Macías F, Magallanes-Quintanar R, Valdez-Cepeda RD, Vázquez-Alvarado R, Olivares-Sáenz E, Gutiérrez-Ornelas E, Murillo-Amador B. Nutritional reference values for Opuntia ficus-indica determined by means of the boundary-line approach. J. Plant Nutr. Soil Sci. 2010, 173, 927-934. doi: 10.1002/jpln.200900147.

[35] Rodríguez-García ME, De Lira C, Hernández-Becerra E, Cornejo-Villegas MA, Palacios-Fonseca AJ, Rojas-Molina I, Muñoz-Torres C. Physicochemical characterization of nopal pads (Opuntia ficus-indica) and dry vacuum nopal powders as a function of the maturation. Plant Foods Hum. Nutr. 2007, 62, 107-112. doi: 10.1007/s11130-007-0049-5.

[36] Rojas-Molina I, Gutiérrez-Cortez E, Bah M, Rojas-Molina A, Ibarra-Alvarado C, Rivera-Muñoz E, Aguilera-Barreiro MDLA. Characterization of calcium compounds in Opuntia ficus-indica as a source of calcium for human diet. J. Chem. 2015, 2015, 710328. doi: 10.1155/2015/710328.

[37] Sáenz C. Cladodes: a source of dietary fiber. J. Prof. Assoc. Cactus. Dev. 1997, 2, 117-123.

[38] Elleuch M, Bedigian D, Roiseux O, Besbes S, Blecker C, Attia H. Dietary fibre and fibre-rich by-products of food processing: Characterisation, technological functionality and commercial applications. Food Chem. 2011, 124, 411-421. doi: 10.1016/j.foodchem.2010.06.077.

[39] López-Cervantes J, Sánchez‑Machado DI, Campas‑Baypoli ON, Sánchez‑Duarte RG. Chemical composition and physicochemical properties of nopal (Opuntia ficus‑indica) flour. Food Sci. Technol. 2011, 31, 676-682. doi: 10.1590/S0101-20612011000300016.

[40] Shoukat R, Cappai M, Pia G, Pilia L. An Updated Review: Opuntia ficus indica (OFI) Chemistry and Its Diverse Applications. Appl. Sci. 2023, 13, 7724. doi: 10.3390/app13137724.

[41] Pourramezan Z, Ghezelbash GR, Romani B, Ziaei S, Hedayatkhah A. Screening and identification of newly isolated cellulose-degrading bacteria from the gut of xylophagous termite Microcerotermes diversus (Silvestri). Mikrobiologiia 2012, 81, 796-802. doi: 10.1134/S0026261712060124.

[42] Rasi RPM, Mahalingam PU. Screening and partial characterization of cellulose degrading bacteria from decayed sawdust. Int. J. Sci. Res. 2014, 3, 957-960.

[43] Bernabé-Salomon LA. Identificación molecular de bacterias cultivadas y no cultivadas asociadas a la rizósfera de Opuntia ficus-indica (L.) Mill. (Cactaceae) en ecosistemas áridos; Bachelor Thesis, Universidad Nacional de Tumbes, Tumbes, Perú, 2016.

[44] Singh R, Kumar M, Mittal A, Mehta PK. Microbial enzymes: industrial progress in 21st century. 3 Biotech. 2016, 6, 174. doi: 10.1007/s13205-016-0485-8.

[45] Dhiman SS, Sharma J, Battan B. Industrial applications and future prospects of microbial xylanases: a review. BioResources 2008, 3, 1377-1402.

[46] Béguin P, Aubert JP. The biological degradation of cellulose. FEMS Microbiol. Rev. 1994, 13, 25-58. doi: 10.1111/j.1574-6976.1994.tb00033.x.

[47] Medina-Torres L, Brito-De La Fuente E, Torrestiana-Sanchez B, Katthain R. Rheological properties of the mucilage gum (Opuntia ficus-indica). Food Hydrocoll. 2000, 14, 417-424. doi: 10.1016/S0268-005X(00)00015-1.

[48] Song W, Lagmay VQ, Jeong B-G, Jung J. Changes in physicochemical and functional properties of Opuntia humifusa during fermentation with cellulolytic enzyme and lactic acid bacteria. LWT-Food Sci. Technol. 2022, 159, 113192. doi: 10.1016/j.lwt.2022.113192.

[49] Malik WA, Javed S. Biochemical characterization of cellulase from Bacillus subtilis strain CD001 and its effect on digestibility and structural modifications of lignocellulose-rich biomass. Front. Bioeng. Biotechnol. 2021, 9, 800265. doi: 10.3389/fbioe.2021.800265.

[50] López-Domínguez CM, Ramírez-Sucre MO, Rodríguez-Buenfil IM. Enzymatic hydrolysis of Opuntia ficus-indica cladode by Acinetobacter pittii and alcohol fermentation by Kluyveromyces marxianus: pH, temperature and microorganism effect. Biotechnol. Rep. 2019, 24, e00384. doi: 10.1016/j.btre.2019.e00384.

[51] Mushtaq Q, Ishtiaq U, Joly N, Spalletta A, Martin P. Harnessing Bacillus subtilis QY5 PP784163 for bioethanol production from potato peel waste and nutrient recovery for animal feed: Maximizing resource efficiency. Fermentation 2024, 10, 523. doi: 10.3390/fermentation10100523.

[52] Boulos N N, Greenfield H, Wills RBH. Water holding capacity of selected soluble and insoluble dietary fibre. Int. J. Food Properties. 2000, 3, 231-244. doi: 10.1080/10942910009524629.

[53] Habibi Y, Mahrouz M, Vignon MR. Structural features of pectic polysaccharides from Opuntia ficus-indica cladodes. Carbohydr. Res. 2005, 340, 695-699. doi: 10.1016/j.carres.2004.11.004.

[54] Kim J, Lee H, Park Y, Ra K, Shin K, Yu K, Suh H. Mucilage removal from cactus cladodes (Opuntia humifusa Raf.) by enzymatic treatment to improve extraction efficiency and radical scavenging activity. LWT-Food Sci. Technol. 2013, 51, 337-342.

[55] Ekperigin MM. Preliminary studies of cellulase production by Acinetobacter anitratus and Branhamella sp. Afr. J. Biotechnol. 2007, 6, 28-33.

[56] Bayar N, Friji M, Kammoun R. Optimization of enzymatic extraction of pectin from Opuntia ficus-indica cladodes after mucilage removal. Food Chem. 2018, 241, 127-134. doi: 10.1016/j.foodchem.2017.08.051.